DUBLIN CITY UNIVERSITY

School of Biotechnology
RESEARCH PROJECT (BE492)
Expression of Myosin Heavy
Chain 11 splice variants in
resident vascular stem cells
Bilikis Sulaimon

Supervised by Professor.
Paul Cahill
School of Biotechnology, Dublin City University, Declaration on Plagiarism
Assignment Submission Form
Name and Student Number:
Programme:
Module Code:
Assignment Title:

Bilikis Sulaimon (11301221)

Biotechnology
BE492
Expression of Myosin Heavy Chain 11 splice variants in

Submission Date:

resident vascular stem cells

22/04/2016

I declare that this material, which I now submit for assessment, is entirely my own work and
has not been taken from the work of others, save and to the extent that such work has been
cited and acknowledged within the text of my work. Text that is directly lifted from a
reference source must be in quotation marks and cited. (In scientific writing, quotations are
rarely used and the text should generally be paraphrased i.e., written in ones own words.) I
understand that plagiarism, collusion, and copying are grave and serious offences in the
university and accept the penalties that would be imposed should I engage in plagiarism,
collusion, or copying. I have identified and included the source of all facts, ideas, opinions,
viewpoints of others in the assignment references. Direct quotations from books, journal
articles, internet sources, module text, or any other source whatsoever are acknowledged and
the source cited are identified in the assignment references. This assignment, or any part of it,
has not been previously submitted by me or any other person for assessment on this or any
other course of study.
I have read and understood the referencing guidelines found at
http://www.library.dcu.ie/LibraryGuides/Citing&RefGuide11.pdfhttp://www.library.dcu.ie/Li
braryGuides/Citing&RefGuide11.pdf and/or recommended by the lecturer giving the
assignment.
I understand that all assignments submitted to the School of Biotechnology will be submitted
to the Plagiarism detection software Turnitin.

Name: Bilikis Sulaimon

Date: 22/04/201

Table of Contents
ABSRACT................................................................................................................ 4
Abbreviations......................................................................................................... 4
INTRODUCTION...................................................................................................... 5
MATERIALS AND METHODS.................................................................................... 6
RESULTS............................................................................................................... 11
DISCUSSION......................................................................................................... 29
CONCLUSION....................................................................................................... 30
ACKNOELEDGEMENTS.......................................................................................... 30
REFERENCES........................................................................................................ 31

ABSRACT
The main objective of this study was to investigate the expression of myosin heavy chain
splice variant in resident vascular stem cells with primer binding and sequences alignments,
the wet experiment of this analysis have previously been done resulting to an unsatisfied
result. Myosin heavy chain (MHC) isoform expression was evaluated by reverse transcription
polymerase chain reaction (RT-PCR), agarose gel electrophoresis and sequencing analysis to
clarify a possible link between resident vascular stem cells and the appearance of these
variants. The key question is whether stem cells including these resident vascular stem cells
express any Myosin 11 (Myh11) splice variants before and/or after differentiation. In the wet
experiments when the stem cells were sequenced, bioinformatics revealed the stem cells were
Myh10. How is Myh11 primers generating Myh10 sequences. Myosin heavy chain 11 is a
smooth muscle cell with four transcripts, SM1A, SM1B, SM2A, SM2B. The wet experiment
indicates that by Isolating the RNA in Mouse and Rat, smooth muscle cells were found in
aorta, bladder and intestine using Myh11 primer. Myh11 primers discriminants SM1A,
SM1B, SM2A and SM2B. When Aortic mRNA and Intestine mRNA was converted to cDNA,
Primers bind to either side to generate amplicons, if any of the fragment has inserts, the
fragment would be longer. The long fragments are SMB and SM2. It was found that Aorta is
primary A and Small Intestine is B & 2. The gel was run with an agarose Gel. Aorta gave one
fragment and Intestine gave two fragments. When stem cells were sequenced, the fragment in
Rat was Myh10 and Myh14 in Mouse. In this research Primer binding will illustrate where
the primers are binding to generate the predicted fragments and where the pair of primers
bind. Sequences analysis will convey differences between Myh10 and Myh11.

Abbreviations
Myosin heavy chain (MHC)
Myosin 11 (Myh11)
Reverse transcription polymerase chain reaction (RT-PCR)
Multipotent vascular stem cell (MVSC)

INTRODUCTION
Cardiovascular diseases (CVD) are leading causes of death in Europe. CVD is responsible for
47% of all deaths in Europe. CVD is the main cause of death in women in all European
countries. Ireland in particular has a huge rate of death associated with this disease with 38%
of annual deaths connected to CVD. The main obstruction of this tragic disease is a result of
the blockage the vessel. Plaque build-up and stiffens the vascular smooth muscle cells
(SMCs) within blood vessel wall leading to heart attack, stroke, aneurysm, hypertension,
arteriosclerosis and atherosclerosis.2
Myosin heavy chain 11 is a biomarker for smooth muscle cells and stem cells can be
specialised to stimulate SMC. Studies from many laboratories have shown that Stem cells
have been a huge in impacted in science, it has been providing tremendous advances in the
treatment of serious human diseases1.
A huge controversy has arisen over the years in the determination of the origin of the vascular
Smooth Muscle Cell (SMC) involved with vascular remodelling. Various groups have
presented data illustrating that these cells may be derived from an unknown population of
vascular stem cells8
Studies have shown that there are various methodologies to approach how the SMC express
their variants. A key to understanding SMC differentiation is to identify transcriptional
mechanisms that control expression of genes that are selective and specific for differentiated
SMC. The three methods listed below will be carried out and compared

To analyse whether the resident vascular stem cells derived from rat or mouse aorta

express any Mhy11 splice variants before and/or after differentiation

To verify if the splice variants are generated when the stem cells are induced
The objectives of this research include computationally working out the fragment. Based
on primer binding.
Secondly cluster multi alignment (sequence alignment)
Firstly, Primer Alignment will be done to generated amplicons and fragments, based on a
primers inserted in the algorithm, using two different algorithms. Primer binding will
state the predicted size of the fragment and its sequences of the amplicons, the pair of
Primers bind to the cDNA, they bind at two locations to generate the size and sequences.
It then generates these sequences; SM1, SM2, SMA, SMB. Isoform SM2 &SMB is
longer than Isoform SM1 &SMA due to the extra nucleotides it has. The sequences are
identical, the only difference is an insert between Isoform SM2 &SMB and SM1 & SMA.
There are 21 nucleotides that codes for the insert. An alignment analysis was done on

both fragments, when the insert is there, there are different homology between A and B.
Differences is that B has extra nucleotides in the middle. 90% of SM B will be SMA.
Secondly Sequence Alignment was done, the alignment was done with or without insert,
aligned with Myh11 gene, to show the fragments generated are 100% homologous to the
What is Bioinformatics: Computer Analysis: Computational Biology/ System biology

OBJECTIVE:

To understand what exactly the primers are binding to, the analysis of Myh11 primers
and where Myh11 primers bind in a Myh11 transcript and Myh10 Transcript and a Rat

genome
To understand the fidelity and homology of the Myh11 primers in Rat genome
To know the size of the fragment and the computational sequence of the generated

sequence after binding

To be certain if the primers bind/not bind to Myh10 Transcript at the stem cells

MATERIALS AND METHODS

Primer Binding
Myh11 Primers for MHC-1/2 were
5AAGCAGCTCAAGAGGCAGCTGGA-3 5-AAGGAACAAATGAAGCCTCGTT3
Primers for MHC-A/B were
TACAGAAGCATGCTACAAGATCGT5GCGGGAGGAGTTGTCATTCTTGAC-3
Rat Myh11 Transcript

>MYH11
GGTAGCGCTGACTCTATAAAGCCAGATGTCCGAAGCATACAGAGAGATTTGGACCATCCCAGCCTGGGATCAGT
GTCAGATCCGAGCTCTCCATCCGGTGTTCTCCTGCTAGTCCACCCCAGTAGCAGATCTGGGACCACCAAGCACA
ATGGCACAGAAAGGGCAGCTCAGTGATGATGAGAAGTTCCTCTTTGTGGATAAAAACTTCATAAACAGCCCAAT
GGCCCAGGCCGACTGGGTAGCCAAGAAGCTAGTATGGGTCCCTTCAGAGAAGCAGGGCTTCGAAGCAGCCAGC
ATCAAGGAGGAGAAGGGCGATGAGGTGGTTGTAGAGCTGGTGGAAAACGGAAAGAAGGTCACAGTGGGCAA
AGATGACATCCAAAAAATGAACCCGCCCAAGTTCTCCAAGGTGGAAGACATGGCCGAGCTGACATGCCTCAAT
GAGGCTTCTGTGCTGCACAACCTGAGGGAGCGGTACTTCTCAGGCCTCATCTACACCTACTCGGGCCTCTTCTG
TGTGGTGGTCAACCCCTACAAGCACCTACCCATCTACTCAGAAAAGATTGTGGACATGTACAAGGGCAAGAAG
AGGCATGAGATGCCGCCTCACATCTATGCCATTGCTGATACGGCCTACAGAAGCATGCTACAAGATCGTGAAGA
CCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTG
GCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGGGGGAGCTGGAAAAGCAGCTTCTA
CAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTCAAGAATGACAACTCCTCCCGCTTTGGCA
AGTTCATTCGCATCAACTTTGATGTCACTGGTTACATTGTGGGTGCCAATATCGAAACATATCTTCTGGAAAAGTC
TCGGGCTATTCGACAGGCTAGGGACGAGAGAACGTTCCACATCTTCTACTATCTGATCGCTGGAGCCAAGGAGA
AGATGAGAAATGACCTACTTTTGGAGAGCTTCAACAGCTACACATTTTTATCCAATGGCTTTGTGCCCATCCCAG
CTGCACAAGATGATGAGATGTTCCAGGAGACACTAGAGGCCATGTCCATCATGGGCTTCAGTGAAGAGGAACA
GCTAGCCATCTTGAAGGTAGTATCATCCGTCCTTCAGCTTGGAAACATTGTCTTCAAGAAGGAGCGAAACACAG
ACCAGGCATCCATGCCTGATAACACAGCTGCTCAGAAAGTTTGCCACCTTGTGGGGATTAATGTGACAGATTTC
ACCAGAGCCATCCTGACCCCACGTATAAAAGTTGGACGGGATGTGGTGCAGAAAGCTCAGACCAAAGAACAGG
CTGACTTTGCCATTGAGGCCTTAGCCAAGGCCACCTATGAGCGCCTTTTCCGATGGATTCTCAGCCGTGTGAACA

AAGCCTTGGATAAGACCCATCGTCAGGGGGCCTCCTTCCTGGGCATTCTGGATATTGCTGGGTTTGAAATCTTTG
AGGTAAACTCCTTCGAGCAGCTGTGCATCAACTACACCAATGAGAAGTTGCAGCAGCTGTTCAATCACACCATG
TTCATCCTGGAGCAGGAAGAGTACCAACGAGAGGGCATCGAGTGGAACTTCATCGACTTTGGCCTGGACCTAC
AGCCTTGTATTGAACTGATTGAGCGGCCGAACAACCCTCCTGGTGTGCTGGCCCTGCTGGATGAAGAATGCTGG
TTCCCCAAAGCTACGGACAAGTCTTTTGTGGAGAAGCTGTGCTCAGAGCAGGGCAATCACCCCAAATTTCAGA
AGCCCAAGCAGCTAAAGGACAAAACAGAGTTCTCCATCATCCACTATGCTGGGAAGGTGGACTATAATGCAAGT
GCGTGGCTGACCAAGAACATGGACCCGCTAAATGATAATGTGACCTCACTCCTCAATGCCTCCTCGGACAAGTT
TGTGGCCGACCTGTGGAAGGATGTGGACCGCATTGTGGGGTTGGACCAGATGGCCAAGATGACTGAGAGCTCA
CTGCCCAGTGCCTCCAAGACCAAAAAGGGCATGTTCCGCACCGTGGGCCAGCTCTACAAAGAGCAGCTGGGGA
AGCTGATGACCACACTGCGCAACACCACGCCTAACTTTGTGCGCTGCATCATCCCCAACCATGAAAAGAGGTCT
GGCAAGCTGGATGCATTTCTAGTTCTGGAGCAGCTGCGGTGCAATGGTGTGTTGGAAGGCATCCGCATCTGCCG
TCAGGGCTTCCCCAACAGGATTGTCTTCCAAGAGTTCCGGCAACGCTACGAGATCCTGGCAGCGAACGCCATCC
CCAAAGGCTTCATGGACGGAAAGCAAGCCTGCATTCTCATGATCAAAGCCCTTGAACTTGACCCCAATCTGTAC
AGGATTGGGCAGAGCAAAATCTTCTTTCGTACGGGAGTCCTGGCCCACCTAGAGGAAGAACGAGACCTGAAGA
TTACTGATGTCATCATGGCTTTCCAGGCAATGTGTCGTGGCTACCTGGCCAGAAAAGCCTTCACCAAGAGGCAG
CAACAGTTAACAGCCATGAAGGTGATCCAGAGGAACTGCGCTGCCTACCTTAAGCTCCGCAACTGGCAATGGT
GGAGGCTCTTCACTAAAGTAAAGCCGCTGCTCCAGGTGACGCGGCAGGAGGAGGAGATGCAGGCCAAGGAGG
AGGAGATGCAGAAGATCAAGGAGCGGCAGCAGAAGGCAGAGTCTGAGCTGAAGGAGCTGGAGCAAAGACAC
ACTCAGCTGGCTGAGGAGAAGACCCTGCTGCAGGAGCAGTTGCAGGCAGAGACAGAGCTGTATGCTGAGGCT
GAAGAGATGCGGGTCCGCTTGGCAGCGAAGAAGCAGGAGCTAGAGGAGATCCTCCATGAGATGGAGGCCCGC
TTGGAGGAGGAGGAGGACCGGAGCCAACAACTGCAGGCTGAGAGGAAGAAGATGGCTCAGCAGATGCTGGA
CCTGGAGGAGCAACTGGAGGAGGAAGAAGCTGCCAGACAGAAACTACAGCTAGAGAAGGTCACAGCTGAGG
CCAAGATCAAGAAACTGGAGGATGACATCTTGGTTATGGATGACCAGAACAATAAGCTTTCCAAAGAAAGAAA
ACTCCTTGAAGAGAGGGTCAGCGACTTGACAACCAACCTAGCAGAAGAGGAAGAAAAGGCTAAAAACCTCAC
AAAACTAAAGAGCAAGCATGAGTCCATGATCTCAGAGCTAGAGGTGAGGCTGAAGAAAGAGGAGAAGAGCCG
GCAGGAGCTGGAGAAACTCAAGAGGAAACTGGAGGGTGATGCCAGTGACTTCCACGAGCAGATCGCTGACCT
GCAGGCCCAGATTGCAGAGCTCAAGATGCAGCTGGCAAAGAAAGAGGAAGAGCTACAGGCAGCTCTAGCCAG
GCTCGATGAAGAGATCACCCAGAAAAACAATGCCCTAAAGAAGATTCGAGAGCTGGAGGGCCATGTCTCAGAC
CTACAGGAGGACCTGGACTCAGAGCGAGCTGCTAGGAACAAGGCTGAGAAACAGAAGCGAGACCTGGGGGA
GGAACTGGAGGCGCTCAAGACAGAGCTGGAAGATACACTGGACAGCACAGCTACCCAGCAGGAGCTCAGAGC
CAAGAGGGAACAGGAAGTGACAATGCTGAAGAAGGCCCTGGATGAAGAGACGCGATCCCATGAGGCCCAGGT
CCAGGAGATGAGGCAGAAGCACACACAGGCAGTGGAGGAACTCACAGAGCAGCTGGAACAGTTCAAAAGGG
CCAAGGCAAACCTGGATAAAAGCAAGCAGACGCTGGAGAAGGAGAACGCAGACCTGGCTGGGGAGCTGCGTG
TCCTGGGCCAGGCAAAGCAGGAGGTAGAGCACAAGAAGAAGAAGCTGGAGGGGCAGCTGCAGGAGCTGCAG
TCCAAGTGCAGTGATGGGGAGCGTGCCCGGACTGAGCTCAGTGACAAGGTCCACAAACTACAGAATGAAGTG
GAGAGTGTCACAGGCATGCTCAATGAGGCAGAAGGCAAGGCCATTAAACTGGCCAAGGAAGTGGCTTCCCTTG
GATCTCAACTTCAGGACACCCAAGAGCTGCTTCAAGAAGAAACCCGGCAGAAGCTCAATGTGTCCACCAAGCT
GCGCCAGTTGGAAGATGAGAGGAACAGCCTGCAGGACCAGCTGGATGAGGAGATGGAAGCTAAGCAAAACCT
TGAGCGCCATGTCTCCACCTTGAACATTCAGCTCTCAGACTCTAAGAAGAAGCTGCAGGACCTTGCAAGCACTA
TTGAGGTCATGGAGGAGGGGAAGAAGAGGTTGCAGAAAGAGATGGAGGGTCTCGGCCAGCAGTATGAGGAGA
AGGCCGCTGCCTATGACAAACTGGAGAAAACTAAGAACAGGCTTCAGCAGGAGCTAGATGACTTGGTCGTGGA
CTTGGACAACCAGCGGCAACTGGTATCCAATCTGGAAAAGAAGCAGAAGAAATTCGATCAGTTGTTAGCTGAG
GAAAAGAACATCTCCTCCAAGTATGCGGATGAGAGAGACCGAGCTGAAGCAGAGGCCAGGGAAAAGGAAACG
AAGGCTTTGTCTCTGGCCCGGGCCCTGGAGGAAGCCCTGGAAGCCAAAGAGGAACTGGAAAGGACCAACAAG
ATGCTCAAGGCCGAGATGGAAGACCTGGTCAGCTCCAAGGATGACGTGGGCAAGAATGTGCACGAGCTGGAG
AAGTCCAAACGAGCCCTGGAGACCCAGATGGAAGAGATGAGAACTCAGCTAGAGGAGCTGGAGGACGAGCTG
CAGGCCACTGAAGACGCCAAGCTGCGGCTGGAGGTCAACATGCAGGCCCTCAAGGGCCAGTTTGAACGAGAT
CTCCAGGCTCGGGATGAGCAGAATGAGGAGAAGAGGAGGCAGCTACAGCGGCAGCTGCATGAGTATGAGACA
GAACTGGAAGATGAACGGAAGCAAAGGGCTCTGGCGGCAGCAGCCAAGAAGAAGCTGGAAGGGGATCTGAA
AGACCTGGAACTCCAGGCTGACTCAGCCGTCAAAGGGAGGGAGGAAGCCATCAAGCAGCTTCGAAAACTGCA
GGCTCAGATGAAGGACTTCCAAAGAGAGCTGGATGATGCCCGTGCCTCCAGAGATGAGATCTTTGCCACCTCA
AAAGAGAATGAGAAGAAAGCCAAGAGTCTGGAGGCAGAGCTCATGCAGCTCCAGGAGGACCTGGCAGCAGCT
GAGAGAGCTCGCAAGCAAGCTGACCTGGAGAAGGAGGAGCTGGCTGAGGAGCTGGCTAGCAGCTTATCAGGA
AGGAATACACTGCAGGATGAGAAGCGCCGCCTGGAGGCAAGGATCGCCCAGCTGGAGGAGGAGCTGGAGGAA
GAGCAGGGCAACATGGAGGCCATGAGTGACAGAGTCCGCAAGGCCACATTGCAGGCTGAGCAACTGAGCAAT
GAGCTGGTCACTGAGCGCAGTGCAGCTCAGAAGAATGAGAGTGCACGGCAACAGCTGGAGCGCCAGAACAAG
GAACTCCGAAGCAAGTTGCAGGAGGTAGAAGGTGCTGTCAAAGCCAAGCTTAAGTCCACTGTTGCGGCACTAG
AGGCCAAGATTGTGCAGCTGGAGGAGCAGATTGAACAAGAGGCCAGAGAAAAACAGGCAGCCACCAAGTTGC
TGAAGCAAAAGGACAAGAAGCTAAAGGAGGTCCTACTGCAGGTGGAGGATGAGCGCAAGATGGTGGAGCAGT
ACAAGGAGCAGGCAGAGAAAGGAAACACCAAGGTCAAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGA
GGAATCCCAGCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGA
GGCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCATTTGTTCCTTCC
AGAAGGGCTGGAGGCCGTAGAGTTATTGAAAACACAGATGGTTCTGAAGAAGAAATGGATGCTCGGGACTCAG
ACTTCAATGGAACCAAAGCCAGTGAATAAATTCAGTTGGACACTATGTCAGGGAAAACAAAATACCAAATGAC
AGCAAAGCCCAGCAGGCTGATTAGCATTTGTCTCCATTCGTTCTCAAGTCATAGACATTACCCCACCTCTCACCA
GGAGTCAACCACAGCCCTGCACAAAGGATGTGAATTTAAGTTTTTAAATGTGCTACCAGAGAAAATCCAGATAG
ATGCAGGTCGGGAGCTGCAGTCGCCAGTGCCTTTGTTTACATGAAATGGAACGTTACGTGAATGGGTGCCCCAG
CCCGCAACCCGCTCCCAACCATCAACTGCCTGACCTTCCAGTTTGGGAGGAAAACCTGTTTGAAAATTCCCCTC
CCTTTCCTGCCTGCACACCTCAGACACACAGTTCACTAAGACCAAGTGACTTTTTCAGTCATCTGAATTAAGGA
AGAGCAGAGCGAGCGCACCGCCTTATTTGCACCCACGGACACTTTCACGAATAAAAGGTTTTAAGTCACGAGA

Rat MYH10 Transcript

NCBI Reference Sequence: NM_031520.1, myosin, heavy chain 10, non-muscle (Myh10), mRNA
ATGGCCCAGAGAACTGGACTGGAGGATCCCGAGAGGTATCTCTTTGTGGACAGGGCTGTCATCTACAACCCTGCCACTCAAGC
TGACTGGACAGCTAAAAAGCTGGTGTGGATTCCATCTGAACGCCATGGTTTTGAGGCAGCTAGTATTAAAGAAGAACGGGGCG
ATGAGGTTATGGTGGAGCTGGCAGAGAACGGGAAGAAAGCGATGGTCAACAAAGATGACATTCAGAAGATGAACCCGCCGAA
GTTCTCCAAGGTGGAGGACATGGCAGAGCTCACGTGCTTGAACGAAGCCTCTGTTTTACACAATCTGAAGGATCGCTACTATT
CAGGACTCATCTATACTTACTCGGGACTCTTCTGTGTGGTGATAAATCCCTACAAGAACCTTCCGATTTACTCTGAGAATATTATT
GAGATGTACAGAGGGAAGAAGCGCCATGAGAAGCCGCCACACATCTACGCCATATCAGAGTCTGCCTACAGATGCATGCTTCA
AGATCGTAAGGACCAGTCGATTCTTTGCACGGGTGAATCAGGTGCCGGGAAGACAGAAAATACAAAGAAAGTCATTCAGTAC
CTTGCCCACGTTGCTTCTTCACACAAAGGAAGAAAGGACCACAATATTCCTGGGGAACTTGAACGGCAGCTTTTACAAGCAAA
TCCAATTCTGGAATCATTTGGAAATGCGAAGACTGTGAAAAATGATAACTCATCTCGCTTTGGCAAGTTTATCCGGATCAACTTT
GATGTAACCGGCTATATTGTTGGGGCCAACATTGAAACATACCTTCTGGAAAAGTCCCGTGCTGTTCGTCAAGCTAAAGATGAA
CGCACGTTTCATATCTTTTATCAGTTGCTCTCTGGAGCAGGGGAACACCTGAAGTCCGACTTACTCCTGGAAGGCTTTAATAAC
TACAGATTCCTCTCCAACGGCTACATCCCTATTCCTGGACAGCAAGACAAGGATAACTTTCAGGAGACCATGGAAGCCATGCAC
ATAATGGGCTTCTCTCACGAAGAGATCCTCTCAATGCTTAAAGTCGTGTCTTCAGTGCTGCAGTTTGGAAACATCTCTTTCAAA
AAGGAGAGGAACACTGACCAAGCTTCCATGCCCGAGAACACAGTTGCACAGAAACTCTGCCACCTGCTTGGGATGAATGTGA
TGGAGTTCACTCGGGCTATCCTCACGCCCAGGATCAAAGTTGGCCGAGATTATGTACAGAAAGCCCAGACCAAAGAGCAGGC
AGATTTTGCAGTGGAAGCATTGGCAAAGGCTACCTATGAGCGGTTGTTTCGCTGGCTCGTTCACCGCATCAATAAGGCGCTGGA
TAGGACCAAACGCCAGGGAACGTCTTTCATTGGGATCCTGGATATTGCTGGTTTTGAAATTTTTGAGCTGAACTCCTTTGAACA
ACTATGCATCAACTACACCAATGAGAAGCTGCAGCAGCTGTTCAACCACACCATGTTCATCCTGGAGCAGGAGGAGTACCAGC
GAGAGGGCATCGAGTGGAACTTCATCGACTTTGGTCTCGACTTGCAGCCCTGCATCGACCTGATAGAGAGACCTGCCAATCCC
CCTGGTGTGCTGGCCCTCCTGGATGAAGAATGCTGGTTCCCCAAAGCCACAGACAAAACGTTTGTTGAGAAGCTGGTTCAGG
AGCAAGGTTCCCACTCCAAGTTTCAGAAACCACGGCAACTGAAAGACAAAGCCGACTTCTGCATCATCCACTATGCAGGGAA
GGTGGACTATAAGGCGGATGAGTGGCTGATGAAGAACATGGACCCCCTGAATGACAACGTGGCCACCCTCCTGCACCAGTCAT
CAGACAGATTTGTCGCCGAGCTTTGGAAGGATGTGGACCGAATTGTAGGTCTGGATCAAGTCACTGGCATGACTGAGACAGCT
TTTGGCTCTGCATATAAAACCAAGAAGGGCATGTTCCGCAACGTCGGGCAGCTCTACAAAGAGTCTCTCACCAAGCTCATGGC
AACCCTCCGAAACACCAACCCCAACTTCGTACGCTGCATCATTCCAAATCACGAGAAGCGGGCTGGAAAACTGGATCCACATC
TTGTGCTGGATCAGCTCCGCTGTAACGGTGTCCTGGAAGGAATCAGGATCTGTCGCCAGGGATTCCCCAACCGGATAGTTTTCC
AGGAGTTCAGACAGAGATATGAGATCCTAACTCCCAACGCTATTCCCAAAGGCTTCATGGATGGCAAACAGGCCTGTGAGCGA
ATGATCCGAGCTTTAGAACTGGACCCAAACCTGTATAGAATTGGACAGAGCAAGATATTTTTCCGAGCTGGAGTTCTGGCGCAC
TTAGAGGAAGAAAGAGATTTAAAAATCACTGATATAATCATCTTTTTCCAAGCTGTATGCAGAGGCTACCTAGCCAGAAAGGCC
TTTGCCAAGAAACAGCAGCAGCTAAGTGCCTTAAAGGTCTTGCAGCGGAACTGTGCGGCGTACCTGAAGCTGCGACACTGGC
AGTGGTGGCGCGTCTTCACAAAGGTGAAGCCTCTCCTCCAAGTGACCCGCCAGGAGGAGGAGCTCCAGGCTAAAGATGAGGA
GCTGCTGAAGGTGAAAGAGAAGCAGACAAAAGTGGAAGGGGAACTTGAGGAGATGGAACGGAAGCACCAGCAGCTGCTGG
AGAGAAGAATATCCTGGCAGAACAGCTGCAGGCCGAGACTGAGCTCTTTGCTGAAGCAGAGGAGATGAGAGCAAGACTCGCT
GCCAAGAAGCAGGAACTGGAGGAGATCCTCCATGACCTGGAGTCCAGGGTGGAGGGAGAGGAAGAGCGAAACCAGATCCTG
CAGAACGAGAAGAAGAAGATGCAAGCGCACATTCAGGATCTAGAAGAGCAACTGGATGAGGAGGAGGGGGCGCGGCAAAA
GCTGCAGCTGGAGAAGGTGACCGCGGAGGCTAAAATCAAGAAGATGGAAGAGGAGGTTCTGCTTCTCGAGGACCAGAATTCC
AAATTTATCAAAGAAAAGAAACTCATGGAAGATCGAATTGCTGAGTGTTCCTCTCAGCTGGCTGAAGAGGAAGAAAAGGCAA
AAAACTTAGCCAAAATCAGGAATAAGCAAGAAGTGATGATCTCGGATTTAGAAGAACGCTTGAAGAAGGAGGAGAAAACACG
GCAGGAACTGGAAAAGGCCAAACGGAAACTGGACGGTGAAACAACTGATCTGCAGGACCAGATCGCCGAGTTGCAAGCACA
GGTCGATGAGCTCAAAGTCCAGTTGACCAAGAAGGAGGAGGAGCTTCAGGGGGCGCTGGCCAGAGGAGATGATGAGACGCT
GCACAAGAATAATGCGCTTAAAGTTGCGCGGGAGCTGCAGGCCCAAATCGCCGAGCTCCAGGAAGACTTTGAGTCTGAAAAG
GCTTCAAGGAACAAGGCTGAGAAACAAAAACGGGACCTGAGTGAGGAGCTGGAAGCTCTAAAGACAGAACTGGAGGACAC
CCTAGACACCACAGCAGCTCAGCAGGAACTCCGCACAAAGCGGGAGCAGGAAGTAGCTGAGCTGAAGAAGGCTCTTGAGGA
TGAAACTAAGAACCACGAAGCTCAGATCCAGGACATGAGACAGAGGCACGCCACGGCACTGGAGGAACTCTCTGAGCAACT
GGAACAGGCTAAGAGGTTCAAAGCCAATCTGGAGAAGAACAAACAGGGCCTGGAGACGGACAACAAGGAGCTGGCGTGTG
AGGTGAAGGTGCTGCAGCAAGTCAAGGCTGAGTCAGAGCACAAGAGGAAGAAGCTCGATGCCCAGGTCCAGGAGCTCCACG
CCAAGGTGTCAGAAGGTGACAGGCTCAGGGTAGAACTGGCCGAGAAAGCAAACAAGCTACAGAATGAGCTGGATAATGTCTC
AACCCTGCTGGAAGAAGCCGAGAAGAAAGGAATGAAGTTCGCTAAGGACGCCGCAGGTCTCGAGTCTCAGCTACAGGACAC
ACAGGAGCTCCTTCAGGAGGAGACGCGGCAGAAACTGAACCTGAGCAGTCGGATCCGGCAGCTGGAGGAGGAGAAGAACA
GCCTTCAGGAGCAGCAGGAGGAGGAAGAGGAGGCCAGGAAGAACTTGGAAAAGCAGGTGTTGGCCCTGCAGTCCCAGCTG
GCTGACACCAAGAAGAAAGTGGACGATGACCTGGGGACAATTGAGGGTTTGGAAGAAGCCAAGAAGAAACTGCTTAAGGAC
GTGGAGGCACTGAGTCAGCGACTGGAGGAAAAGGTCCTAGCATATGACAAGCTGGAGAAGACCAAGAACCGGCTGCAACAG
GAGCTGGATGACCTGACGGTGGACCTGGACCACCAGCGCCAGATCGTCTCCAACTTGGAGAAGAAACAGAAGAAGTTTGACC
AGCTGTTGGCAGAAGAGAAGGGCATCTCTGCTCGCTATGCAGAAGAACGGGACCGGGCTGAAGCTGAGGCCAGAGAGAAAG
AAACCAAGGCGCTCTCCCTGGCGCGGGCCCTGGAGGAGGCCTTGGAGGCCAAGGAGGAATTCGAGAGGCAGAATAAGCAGC
TTCGGGCAGACATGGAAGACCTCATGAGCTCGAAAGATGATGTGGGGAAGAACGTCCACGAGCTGGAGAAGTCCAAGCGGG
CCCTGGAGCAGCAGGTGGAGGAGATGAGGACCCAGCTGGAGGAGCTGGAGGACGAGTTGCAGGCCACTGAGGACGCCAAA
CTCCGCCTGGAAGTCAACATGCAGGCCATGAAGGCCCAGTTTGAGAGGGACCTGCAAACCAGAGATGAGCAGAATGAAGAG
AAGAAGCGGCTGCTGCTCAAGCAGGTACGGGAGCTCGAGGCAGAGCTGGAGGATGAGCGGAAGCAGCGGGCACTTGCTGTG
GCTTCAAAGAAGAAGATGGAGATAGACCTGAAGGACCTGGAGGCTCAGATCGAGGCCGCCAACAAGGCCCGGGATGAAGTG
ATCAAGCAGCTTCGCAAGCTTCAGGCACAGATGAAGGATTACCAGCGTGAACTAGAAGAAGCTCGAGCATCTAGAGATGAGA
TTTTTGCTCAATCCAAAGAAAGTGAAAAGAAACTGAAGAGTCTAGAAGCAGAAATCCTCCAGTTGCAAGAGGAGCTGGCCTC
ATCTGAGCGAGCCCGCAGACACGCAGAGCAGGAGCGAGATGAGCTGGCTGACGAGATTGCCAACAGCGCCTCTGGGAAGTC
CGCTCTGCTGGATGAGAAGCGGCGCCTGGAAGCCCGGATCGCGCAGCTGGAGGAGGAGCTAGAAGAGGAGCAGAGTAACAT
GGAGCTGCTCAATGACCGCTTCCGCAAGACCACGCTCCAGGTGGACACCCTGAACACAGAGTTGGCAGCGGAGCGCAGCGCT
GCCCAGAAGAGCGACAATGCCCGCCAGCAGCTGGAGCGACAAAACAAGGAGCTGAAGGCCAAGCTGCAGGAGCTGGAGGG
GGCGGTCAAGTCCAAGTTCAAAGCCACCATCTCAGCCCTGGAAGCCAAGATTGGGCAGCTGGAGGAACAGCTGGAGCAGGA
AGCCAAGGAACGCGCAGCTGCCAACAAACTATCCGTCGGACAGAGAAGAAACTGAAAGAGATCTTCATGCAGGTCGAAGAC
GAGCGCCGGCATGCGGATCAGTATAAGGAGCAGATGGAGAAAGCCAATGCCAGGATGAAGCAGCTTAAACGCCAGCTGGAAG
AAGCTGAGGAGGAGGCTACACGTGCCAATGCATCTCGGCGTAAACTCCAAAGGGAACTGGATGATGCCACCGAGGCCAATGA
GGGCCTGAGCCGTGAGGTCAGCACTCTCAAGAACCGGCTTAGGCGGGGCGGCCCAATCAGCTTTTCTTCAAGCCGATCTGGCC
GGCGCCAGCTACACATTGAGGGGGCATCGCTAGAGCTGTCAGATGACGACACAGAAAGCAAGACCAGTGATGTCAATGAGAC
ACAGCCACCCCAGTCGGAATAG

Algorithms
MFEprimer-2.0 for Primer alignment

http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/
MFEprimer-2.0 is a newer algorithm to replace NCBI BLAST program for primer
binding sites searching.
National Centre of Biotechnology Centre
Sequence Alignment:
Reverse complement of DNA: http://www.bioinformatics.org/sms/rev_comp.html
Multiple Sequences alignment:
http://multalin.toulouse.inra.fr/multalin/
Multiple Sequences alignment:
http://www.ebi.ac.uk/Tools/msa/clustalw2/
Methods:
1. Primer Binding

Using the databases listed above for primer binding, the given primers was aligned to
the Myh11 Transcript to generate predicted fragments.

The aim of this analysis is to know if the Myh11primers bind to the Myh11 transcript, Myh10
transcript or Myh10 genome. i.e Primer fidelity, also to illustrates what the primers are
binding to, also to know the computational predicted fragments, sequences and the sizes.

RESULTS
After using two different databases, these sequence binded at the temperature the experiments
were carried out. Sequence binded only at the Myh11 transcript and not anywhere else on the
rat genome and definitely not Myh10. When primers bind to the transcript, it theoretically
generates fragments of the following amount of base pair.
MFEprimer-2.0 Report:
Query
ID
Seq1

Sequences 5-3

Lengt

GC%

Tm

AAGCAGCTCAAGAGGCAGCTGG

h
23

56.5

67.1

Seq2

A
AAGGAACAAATGAAGCCTCGTT

22

40.9

59.4

Three potential amplicons were found. Predicted fragments were Rattus norvegicus
myosin, heavy chain 11, smooth muscle, transcript variant 3 (Myh11), mRNA.
PPC = 96.9%
Size = 178 bps
GC content = 57.3%
Fp: Tm = 67.1 (oC), G = -27.1 (kcal/mol), -3.1 (kcal/mol)
Rp: Tm = 59.4 (oC), G = -22.5 (kcal/mol), -3.1 (kcal/mol)

Isoform 1 (SM1): 178 bp

AAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAAT
GCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAG
GCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAG
GCTTCATTTGTTCCTT
Isoform 2 (SM2): 217 bp
AAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAAT
GCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAG
GCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGAGGGCCCCCCCCA
CAAGAAACTTCGCAGTGAGCCACCAGGAGAGGAAACGAGGC

MHC A/B
5TACAGAAGCATGCTACAAGATCGT3and5GCGGGAGGAGTTGTCATTCTTGAC3
Using MFEprimer-2.0 Report, Query ID in
Query

Sequences 5-3

Lengt

GC%

Tm

ID
Seq1
Seq2

TACAGAAGCATGCTACAAGATCGT
GCGGGAGGAGTTGTCATTCTTGA

h
24
24

41.7
54.2

60.7
64.3

C
3 potential amplicons were found
PPC = 100%
Size = 261 bps
GC content = 49.8%
Fp: Tm = 60.7 (oC), G = -24.4 (kcal/mol), 3'G = -3.1 (kcal/mol)
Rp: Tm = 64.3 (oC), G = -26.6 (kcal/mol), 3'G = -3.1 (kcal/mol)
Binding sites: 580(24/24) .840(24/24)

Isoform A (SMA): 240 bp

TACAGAAGCATGCTACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGGTGAGT
CTGGAGCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGG
TGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGGGGGAGCTGGAAA
AGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGT
CAAGAATGACAACTCCTCCCGC
Isoform B (SMB): 261 pb
TACAGAAGCATGCTACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGGTGAGT
CTGGAGCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGG
TGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGCAAGGCCCATCTTT
TGCCTACGGGGAGCTGGAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGC
TTTCGGCAATGCAAAAACTGTCAAGAATGACAACTCCTCCCGC

Sequences Alignments
1. Myh11 Transcript against Predicted Fragments
Myh11 Transcript against SMA
Myh11T

GCCTCACATCTATGCCATTGCTGATACGGCCTACAGAAGCATGCTACAAGATCGTGAAGA

SMA

-------------------------------TACAGAAGCATGCTACAAGATCGTGAAGA
*****************************

Myh11T

CCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGT

SMA

CCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGT
************************************************************

Myh11T

CATACAGTATTTGGCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCAC

SMA

CATACAGTATTTGGCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCAC
************************************************************

Myh11T

GGGGGAGCTGGAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGC

SMA

GGGGGAGCTGGAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGC
************************************************************

Myh11T

AAAAACTGTCAAGAATGACAACTCCTCCCGCTTTGGCAAGTTCATTCGCATCAACTTTGA

SMA

AAAAACTGTCAAGAATGACAACTCCTCCCGC----------------------------*******************************

Myh11 Transcript against SMB

Myh11T

GCCTCACATCTATGCCATTGCTGATACGGCCTACAGAAGCATGCTACAAGATCGTGAAGA

SMB

-------------------------------TACAGAAGCATGCTACAAGATCGTGAAGA
*****************************

Myh11T

CCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGT

SMB

CCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGT
************************************************************

Myh11T

CATACAGTATTTGGCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCAC

SMB

CATACAGTATTTGGCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCAC
************************************************************

Myh11T

G---------------------GGGGAGCTGGAAAAGCAGCTTCTACAGGCAAACCCAAT

SMB

GCAAGGCCCATCTTTTGCCTACGGGGAGCTGGAAAAGCAGCTTCTACAGGCAAACCCAAT
*

**************************************

Myh11T

CCTGGAGGCTTTCGGCAATGCAAAAACTGTCAAGAATGACAACTCCTCCCGCTTTGGCAA

SMB

CCTGGAGGCTTTCGGCAATGCAAAAACTGTCAAGAATGACAACTCCTCCCGC-------****************************************************

Myh11 Transcript against SM1

Myh11T

GGCAGAGAAAGGAAACACCAAGGTCAAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGA

SM1

-------------------------AAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGA
***********************************

Myh11T

GGAATCCCAGCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCAC

SM1

GGAATCCCAGCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCAC

************************************************************

Myh11T

TGAGAGCAATGAGGCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGG

SM1

TGAGAGCAATGAGGCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGG
************************************************************

Myh11T

AAACGAGGCTTCATTTGTTCCTTCCAGAAGGGCTGGAGGCCGTAGAGTTATTGAAAACAC

SM1

AAACGAGGCTTCATTTGTTCCTT------------------------------------***********************

Myh11 Transcript against SM2

Myh11T

AGGCAGAGAAAGGAAACACCAAGGTCAAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGG

SM2

--------------------------AAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGG
**********************************

Myh11T

AGGAATCCCAGCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCA

SM2

AGGAATCCCAGCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCA
************************************************************

Myh11T

CTGAGAGCAATGAGGCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAG

SM2

CTGAGAGCAATGAGGCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGAGGGC
*******************************************************

Myh11T

GAAACGAGGCTTCATTTGTTCCTTCCAGAAGGGCTGGAGGCCGTAGAGTTATTGAAAACA

SM2

CCCCCCCACAAGAAACTTCGCAGTGAGCCACCAGGAGAGGAAA---------CGAGGCTT
*

****

**

Myh11T

CAGATGGTTCTGAAGAAGAAATGGATGCTCGGGACTCAGACTTCAATGGAACCAAAGCCA

SM2

CATTTGTTCCTT-----------------------------------------------**

** * **

2. Myh10 Transcript aligned against Predicted Fragments

Myh10 Transcript aligned against SMA

Myh10T

GCCTACAGATGCATGCTTCAAGATCGTAAGGACCAGTCGATTCTTTGCACGGGTGAATCA

SMA

---TACAGAAGCATGCTACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGGTGAGTCT
****** ******* ********* * ******** ***** ***** ***** **

Myh10T

GGTGCCGGGAAGACAGAAAATACAAAGAAAGTCATTCAGTACCTTGCCCACGTTGCTTCT

SMA

GGAGCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGTGGCATCC
** ** ** ******** ** ************** *****

* **

** ** **

Myh10T

TCACACAAAGGAAGAAAGGACCACAATATTCCTGGGGAACTTGAACGGCAGCTTTTACAA

SMA

TCCCACAAAGGCAAGAAAGACAGCAGCATCACGGGGGAGCTGGAAAAGCAGCTTCTACAG
** ******** *

** ***

**

**

* ***** ** ***

******* ****

Myh10T

GCAAATCCAATTCTGGAATCATTTGGAAATGCGAAGACTGTGAAAAATGATAACTCATCT

SMA

GCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTCAAGAATGACAACTCCTCC
***** ***** *****

* ** ** ***** ** ***** ** ***** ***** **

Myh10T

CGCTTTGGCAAGTTTATCCGGATCAACTTTGATGTAACCGGCTATATTGTTGGGGCCAAC

SMA

CGC--------------------------------------------------------***

Myh10 Transcript aligned against SMB

Myh10T

GCCTACAGATGCATGCTTCAAGATCGTAAGGACCAGTCGATTCTTTGCACGGGTGAATCA

SMB

---TACAGAAGCATGCTACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGGTGAGTCT
****** ******* ********* * ******** ***** ***** ***** **

Myh10T

GGTGCCGGGAAGACAGAAAATACAAAGAAAGTCATTCAGTACCTTGCCCACGTTGCTTCT

SMB

GGAGCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGTGGCATCC
** ** ** ******** ** ************** *****

* **

** ** **

Myh10T

TCACACAAAGGAAGAAAGGACCACAATATTC---------------------CTGGGGAA

SMB

TCCCACAAAGGCAAGAAAGACAGCAGCATCACGCAAGGCCCATCTTTTGCCTACGGGGAG
** ******** *

** ***

**

**

*****

Myh10T

CTTGAACGGCAGCTTTTACAAGCAAATCCAATTCTGGAATCATTTGGAAATGCGAAGACT

SMB

CTGGAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACT
** ***

******* **** ***** ***** *****

* ** ** ***** ** ***

Myh10T

GTGAAAAATGATAACTCATCTCGCTTTGGCAAGTTTATCCGGATCAACTTTGATGTAACC

SMB

GTCAAGAATGACAACTCCTCCCGC-----------------------------------** ** ***** ***** ** ***

Myh10 Transcript aligned against SMA

Myh10T

GAGCGCCGGCATGCGGATCAGTATAAGGAGCAGATGGAGAAAGCCAATGCCAGGATGAAG

SM1

---------------------------------------------------------AAG
***

Myh10T

CAGCTTAAACGCCAGCTGGAAGAAGCTGAGGAGGAGGCTACACGTGCCAATGCATCTCGG

SM1

CAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATGCCAACCGC
***** **

* *********** ** ********

**

******

**

Myh10T

CGTAAACTCCAAAGGGAACTGGATGATGCCACCGAGGCCAATGAGGGCCTGAGCCGTGAG

SM1

AGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGTGAG
* ** ** **

**** ******** ***** ***

******** * ** ********

Myh10T

GTCAGCACTCTCAAGAACCGGCTTAGGCGGGGCGGCCCAATCAGCTTTTCTTCAAGCCGA

SM1

GTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCATTTGTTCCTT----** *

* ******* *

** *** * **

***

* *

Myh10 Transcript aligned against SM1

Myh10T

GAGCGCCGGCATGCGGATCAGTATAAGGAGCAGATGGAGAAAGCCAATGCCAGGATGAAG

SM2

---------------------------------------------------------AAG
***

Myh10T

CAGCTTAAACGCCAGCTGGAAGAAGCTGAGGAGGAGGCTACACGTGCCAATGCATCTCGG

SM2

CAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATGCCAACCGC
***** **

* *********** ** ********

**

******

**

Myh10T

CGTAAACTCCAAAGGGAACTGGATGATGCCACCGAGGCCAATGAGGGCCTGAGCCGTGAG

SM2

AGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGTGAG
* ** ** **

**** ******** ***** ***

******** * ** ********

Myh10T

GTCAGCACTCTCAAGAACCGGCTTAGGCGGGGCGGCCCAATCAGCTTTTCTTCAAGCCGA

SM2

GTGAATGCCCTCAAGAGCAAACTCAGAGGGCCCCCCCCACAAGAAACTTCGCAGTGAGCC
** *

* ******* *

** **

**

****

***

Myh10T

TCTGGCCGGCGCCAGCTACACATTGAGGGGGCATCGCTAGAGCTGTCAGATGACGACACA

SM2

ACCAGGAGA-----------------GGAAACGAGGCTTCATTTGTTCCTT--------*

**

***

***

3. There are no results of the predicted fragments binding to the Myh10 genome
4. Mhy11 Amplicons against Wet Experimental Fragments: The experimental
sequences are partial sequencing SMA is primarily Aorta, SMB is primary intestine.

SMA Transcript against Aorta

SMA

----------TACAGAAGCATGCTACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGG

Aorta

CTTTCTTGGTTACAGAAGCATGCTACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGG
**************************************************

SMA

TGAGTCTGGAGCTGGAAAGACA--------------------------------------

Aorta

TGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGT
**********************

SMA

------------------------------------------------------------

Aorta

GGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGGGGGAGCTGGAAATGCCGGT

SMA

--------------------GAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGTGG

Aorta

GAGTCTGGAGCTGGAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGTGG
****************************************

SMA

CATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGGGGGAGCTGGAAAAGCAGCTTC

Aorta

CATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGAGGGAGCTGGAAAAGCAGCTTC
************************************** *********************

SMA

TACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTCAAGAATGACAACT

Aorta

TACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTCAAGAATGACAACT
************************************************************

SMA

CCTCCCGC-----

Aorta

CCTCCCGCATCTG

********

SMB against Intestine

SMB

TACAGAAGCATGCTACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGGTGAGTCTGGA

R_INT

------------------------------------------------------------

SMB

GCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGTGGCATCCTCC

R_INT

------------------------------------------------------------

SMB

CACAAAGGCAAGAAAGACAGCAGCATCACGCAAGGCCCATCTTTTGCCTACGGGGAGCTG

R_INT

------------------------ATCACGAAAGGCCCATCTTTTGCCTACGGGGAGCTG
****** *****************************

SMB

GAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTC

R_INT

GAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTC
************************************************************

SMB

AAGAATGACAACTCCTCCCGC---------------------------------------

R_INT

AAGAATGACAACTCCTCCCGCCATTCACAACGGTTTACAGAAGCATGCTACAAGATCGTG
*********************

SMB

------------------------------------------------------------

R_INT

AAGACCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGA

SMB

--------------------------

R_INT

AAGTCATACAGTATTTGGCTGTGGTT

SMB against Colon

SMB

TACAGAAGCATGCTACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGGTGAGTCTGGA

R_Col

-------------------------------------------------GTGAGTCTGGA
***********

SMB

GCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGTGGCATCCTCC

R_Col

GCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGTGGCATCCTCC
************************************************************

SMB

CACAAAGGCAAGAAAGACAGCAGCATCACGCAAGGCCCATCTTTTGCCTACGGGGAGCTG

R_Col

CACAAAGGCAAGAAAGACAGCAGCATCACGAGGGAGACAAGATCGTGAAGACCAGTCCAT
******************************

**

SMB

GAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTC

R_Col

TCTGTGCACAG-------GTGAGTCTGGAGCTGGAAAG---------------------***

SMB

AAGAATGACAACTCCTCCCGC

R_Col

---------------------

*****

SMA against Colon

SMA

TACAGAAGCATGCTACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGGTGAGTCTGGA

R_Col

-------------------------------------------------GTGAGTCTGGA
***********

SMA

GCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGTGGCATCCTCC

R_Col

GCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGGTGGCATCCTCC
************************************************************

SMA

CACAAAGGCAAGAAAGACAGCAGCATCACGGGGGAGCTGGAAAAGCAGCTTCTACAGGCA

R_Col

CACAAAGGCAAGAAAGACAGCAGCATCACGAGGGAGACAAGATCGTGAAGACCAGTCCAT
****************************** *****

* *

SMA

AACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTCAAGAATGACAACTCCTCCCGC

R_Col

TCTGTGCACA--GGTGAGTCTGGAGCTGGAAAG--------------------------*

**

** ***

SM1 against Aorta

SM1

AAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATGCCAAC

R_Aorta

-------------------------------------------------TCAATGCCAAC
***********

SM1

CGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGT

R_Aorta

CGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGT
************************************************************

SM1

GAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCATTTGTTCCTT--

R_Aorta

GAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCATTTGTTCCTATG
*********************************************************

SM1

------------------------------------------------------------

R_Aorta

GCCTGTAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATG

SM1

------------------------------------------------------------

R_Aorta

CCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGG

SM1

-------------

R_Aorta

GCCGTGAGGTGAA

SM2 against Aorta

SM2

------------------------------------------------------------

R_Aorta

TCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGG

SM2

------------------------------------------------------------

R_Aorta

CCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCAT

SM2

----------------AAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCA

R_Aorta

TTGTTCCTATGGCCTGTAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCA
*******************************************

SM2

GCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAA

R_Aorta

GCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAA
************************************************************

SM2

TGAGGCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGAGGGCCCCCCCCACA

R_Aorta

TGAGGCCATGGGCCGTGAGGTGAA-----------------------------------************************

SM2

AGAAACTTCGCAGTGAGCCACCAGGAGAGGAAACGAGGCTTCATTTGTTCCTT

R_Aorta

-----------------------------------------------------

SM2 against Colon

SM2

------------------------------------------------------------

R_Col

CTCATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGG

SM2

------------------------------------------------------------

R_Col

CCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCAT

SM2

----------------AAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCA

R_Col

TTGTTCCTACGTGGTGAAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCA
********************************************

SM2

GCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAA

R_Col

GCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAA
************************************************************

SM2

TGAGGCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGAGGGCCCCCCCCACA

R_Col

TGAGGCCATGGGCCGTGAGGTGAAGC---------------------------------************************

SM2

AGAAACTTCGCAGTGAGCCACCAGGAGAGGAAACGAGGCTTCATTTGTTCCTT

R_Col

-----------------------------------------------------

SM1

AAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATGCCAAC

R_Col

-------------------------------------------------CTCATGCCAAC
********

SM1

CGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGT

R_Col

CGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGT
************************************************************

SM1

GAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCATTTGTTCCTT--

R_Col

GAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCATTTGTTCCTACG
*********************************************************

SM1

------------------------------------------------------------

R_Col

TGGTGAAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATG

SM1

------------------------------------------------------------

R_Col

CCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGG

SM1

---------------

R_Col

GCCGTGAGGTGAAGC

SM2

------------------------------------------------------------

R_Col

CTCATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGG

SM2

------------------------------------------------------------

R_Col

CCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCAT

SM2

----------------AAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCA

R_Col

TTGTTCCTACGTGGTGAAGCAGCTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCA
********************************************

SM2

GCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAA

R_Col

GCGCATCAATGCCAACCGCAGGAAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAA
************************************************************

SM2

TGAGGCCATGGGCCGTGAGGTGAATGCCCTCAAGAGCAAACTCAGAGGGCCCCCCCCACA

R_Col

TGAGGCCATGGGCCGTGAGGTGAAGC---------------------------------************************

SM2

AGAAACTTCGCAGTGAGCCACCAGGAGAGGAAACGAGGCTTCATTTGTTCCTT

R_Col

-----------------------------------------------------

SM1 Intestine

SM1

------------------------------------------------------------

Rat_Int

GCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGTGAGGTGAATGC

SM1

------------------------------------------------------AAGCAG

RAT_Int

CCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCATTTGTTCCTTTACCGTAAAGCAG
******

SM1

CTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATGCCAACCGCAGG

RAT_Int

CTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATGCCAACCGCAGG
************************************************************

SM1

AAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGTGAGGTG

RAT_Int

AAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGTGAGGTG
************************************************************

SM1

AATGCCCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCATTTGTTCCTT

RAT_Int

A--------------------------------------------------*

SM2 Intestine

SM2

------------------------------------------------------------

RAT_Int

GCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGTGAGGTGAATGC

SM2

------------------------------------------------------AAGCAG

RAT_Int

CCTCAAGAGCAAACTCAGGAGAGGAAACGAGGCTTCATTTGTTCCTTTACCGTAAAGCAG
******

SM2

CTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATGCCAACCGCAGG

RAT_Int

CTCAAGAGGCAGCTGGAAGAGGCCGAGGAGGAATCCCAGCGCATCAATGCCAACCGCAGG
************************************************************

SM2

AAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGTGAGGTG

RAT_Int

AAGCTGCAGCGGGAGCTGGATGAGGCCACTGAGAGCAATGAGGCCATGGGCCGTGAGGTG
************************************************************

SM2

AATGCCCTCAAGAGCAAACTCAGAGGGCCCCCCCCACAAGAAACTTCGCAGTGAGCCACC

RAT_Int

A----------------------------------------------------------*

SM2

AGGAGAGGAAACGAGGCTTCATTTGTTCCTT

RAT_Int

-------------------------------

Sequence Alignment with SMA&SMB PCR Fragments with MSC

Aorta_SMA&B

TGCCGGTGAGTCTGGAGCTGGAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCT

MSC

------------------------------------------------------------

Aorta_SMA&B

GTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGAGGGAGCTGGAAAAG

MSC

--------------------------------------------------ACTTGAACGG
** ***

Aorta_SMA&B

CAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTCAAGAAT

MSC

CAGCTTTTACAAGCAAATCCAATTCTGGAATCTTTTGGAAATGCGAAGACTGTCAAGAAT
****** **** ***** ***** *****

**** ** ***** ** ************

Aorta_SMA&B

GACAACTCCTCCCGCATCTGCTTTCTTGGTTACAGAAGCATGCTACAAGATCGTGAAGAC

MSC

GACACTCCTCCCGCGATC-CTCCCTCTTCTTACAGAAGCATGCTACAAGATCGTGAAGAC
****

**

***

*******************************

Aorta_SMA&B

CAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGTC

MSC

CAGTCGATTCTG-----------------------------------------------***** ******

Aorta_SMA&B

ATACAGTATTTGGCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACG

MSC

------------------------------------------------------------

Aorta_SMA&B

GGGGAGCTGGAAA

MSC

-------------

AORTA MSC+

Aorta_SMA&B

TGCCGGTGAGTCTGGAGCTGGAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCT

MSC+

------------------------------------------------------------

Aorta_SMA&B

GTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGAGGGAGCTGGAAAAG

MSC+

------------------------------------------------------------

Aorta_SMA&B

CAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGCAAAAACTGTCAAGAAT

MSC+

-----------------------------AGCTTTTGGAAATGCAAAAACTGTCAAGAAT
***** ** * ********************

Aorta_SMA&B

GACAACTCCTCCCGCATCTGCTTTCTTGGTTACAGAAGCATGCTACAAGATCGTGAAGAC

MSC+

GACACTCC-------TCCCGCTGACTGGGTTACAGAAGCATGCTACAAGATCGTG----****

* ***

** ****************************

Aorta_SMA&B

CAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGTC

MSC+

------------------------------------------------------------

Aorta_SMA&B

ATACAGTATTTGGCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACG

MSC+

------------------------------------------------------------

Aorta_SMA&B

GGGGAGCTGGAAA

MSC+

-------------

COLON
Colon_SMA&B

GTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGA----AAGTCATACAGTATTTGGCT

MSC

-----------ACTTGAACGGCAGCTTTTACAAGCAAATCCAATTCTGGAATCTTTTGGA
** *** * ***

****

* *

* * *** *

Colon_SMA&B

GTGGTGGCATCCTCCCACAAAGGCA-------------AGAAAGACAGCAGCATCACGAG

MSC

AATGCGAAGACTGTCAAGAATGACACTCCTCCCGCGATCCTCCCTCTTCTTACAGAAGCA
* *

* * ** * **

* *

Colon_SMA&B

GGAGACAAGATCGTGAAGACCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAG

MSC

TGCTACAAGATCGTGAAGACCAGTCGATTCTG--------------------------*

********************* ******

Colon_SMA&B

GTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGTCATACAGTATTTGGCTGTGG

MSC+

-AGCTTTTGGAAATGCAAAAACTGTCAAGAATGACACTCCTCCCGCTGACTGGGTTACAG
*

* ****

** *** ** *

** *

* *

* * ** *

Colon_SMA&B

TGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGAGGGAGACAAGATCGTGAA

MSC+

AAGCATG--------------------------------------CTACAAGATCGTG-****

***********

Colon_SMA&B

GACCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAG

MSC+

------------------------------------------

Colon_SMA&B

----------------------------------------GTGAGTCTGGAGCTGGAAAG

MVSC

CCACGTTGCTTCTTCACACAAAGGAAGAAAGGACCACAATATTCCTGGGGAACTTGAACG
*

*** ** *** *

Colon_SMA&B

ACAGAGAACACAAAGAA-----AGTCATACAGTATTTGGCTGTGGTG-------------

MVSC

GCAGCTTTTACAAGCAAATCCAATTCTGGAATCATTTGGAAATGCGAAGACTGTCAAGAA
***

****

**

* **

******

**

Colon_SMA&B

---GCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCACGAGGGAGACAAGATCGTGA

MVSC

TGACACTCCTCCCGCTGA----GTGTTTCGATATTACAGAAGCATGCTACAAGATCGTGA
******* *

* *

************

Colon_SMA&B

AGACCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAG-----------------

MVSC

GGACCAGTCGATTCTTTGCACGGGTGAATCAGGTGCCGGGAAGACAGAAAATACAAAGAA
******** ***** ***** ***** ** ** ** ** ***

Colon_SMA&B

-------------------------

MVSC

AGTCATTCAGTACCTTGCCCACGTT

Colon_SMA&B

----------GTGAG---------------------------------------------

MVSC+

GCTGATGCTACTGAGCCCCAGTCTCCTGAGGCAGAAAGCGTCGTTGCCGGAGTGTGGACT
****

Colon_SMA&B

-------------------------TCTGGAGCTGGAAA-GACAGAGAACACAAAGAAAG

MVSC+

TCTCCTCTTCCACCTCTGAGGCAGAGCTAGGGCTGGCACCCTCAGGAGAGCCCCCTTCAT
** * ***** *

***

Colon_SMA&B

TCATACAGTATTTGGCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCA

MVSC+

CCCTTGAGCAGTCAGGTCCCGCATCTTCCTCATACTCTGTCAAGAATGACAACTCCTCCC
* *

** * *

* *

* *****

**

* ****** **** *

Colon_SMA&B

CGAGG-----------GAGACAAGATCGTGA-----------------------------

MVSC+

GCTATTACAGAAGCATGCTACAAGATCGTGGGGGGAAGGTAACATAGTTGGTCAAAGGTC
*

***********

Colon_SMA&B

------------------------------------AGACCAGTCCATTCTGTGCACAGG

MVSC+

ATCAGTGCAGTTTTGCCCTTCCCTTAGGGACAGGGCAAGTCAGTCCAGTGTGTGGTCCTT
*

******* * ****

Colon_SMA&B

TGAGTCTGGAGCTG--------G------------AAAG---------------------

MVSC+

GGAGCCAAGGGGGAAGGCCCAGGGAGTGTCACAGTATAGCAGCAGGAATAATAGTGTGAG
*** *

Colon_SMA&B

---

MVSC+

TTT

* *

* **

Stem cells against Myh11

Myh11_T

GGGGGAGCTGGAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGC

MSC

------ACTTGAACGGCAGCTTTTACAAGCAAATCCAATTCTGGAATCTTTTGGAAATGC
** ***

******* **** ***** ***** *****

**** ** *****

Myh11_T

AAAAACTGTCAAGAATGACAACTCCTCCCGCTTTGGCAAGTTCATTCGCATCAACTTTGA

MSC

GAAGACTGTCAAGAATGACACTCCTCCCGCGATCCTCCCTCTTCTTACAGAAGCATGCTA
** ****************

**

**

Myh11_T

TGTCACTGGTTACATTGTGGGTGCCAATATCGAAACATATCTTCTGGAAAAGTCTCGGGC

MSC

CAAG---------ATCGTGAAGACC----------AGTCGATTCTG-------------** ***

**

*****

Myh11_T

GGGGGAGCTGGAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAATGC

MSC+

---------------------------------------------AGCTTTTGGAAATGC
***** ** *****

Myh11_T

AAAAACTGTCAAGAATGACAACTCCTCCCGCTTTGGCAAGTTCATTCGCATCAACTTTGA

MSC+

AAAAACTGTCAAGAATGACACTCCTCCCGC-TGACTGGGTTACAGAAGCATG-------********************

**

* **

****

Myh11_T

CGAGCTGACATGCCTCAATGAGGCTTCTGTGCTGCACAACCTGAGGGAGCGGTACTTCTC

MVSC

----------------------------------------------CCACGTTGCTTCTT
** * *****

Myh11_T

AGGCCTCATCTACACCTACTCGGGCCTCTTCTGTGTGGTGGTCAACCCCTACAAGCACCT

MVSC

CACACAAAGGAAGAAAG-GACCACAATATTCCTGGGGAACTTGAACGGCAGCTTTTACAA
*

Myh11_T

* *

* ***

* *

* ***

**

ACCCATCTACTCAGAAAAGATTGTGGACATGTACAAGGGCAAGAAGAGGCATGAGATGCC

MVSC

GCAAATCCAATTCTGGAA--------TCATTTGGAAATGCGAAGACTGTCA------AGA
*

*** * *

**

*** *

**

** *

* **

Myh11_T

GCCTCACATCTATGCCATTGCTGATACGGCCTACAGAAGCATGCTACAAGATCGTGAAGA

MVSC

ATGACACTCCTCCCGCTGAGTGTTTCGATATTACAGAAGCATGCTACAAGATCGTGAGGA
***

**

************************** **

Myh11_T

CCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAAGT

MVSC

CCAGTCGATTCTTTGCACGGGTGAATCAGGTGCCGGGAAGACAGAAAATACAAAGAAAGT
****** ***** ***** ***** ** ** ** ** ******** ** ***********

Myh11_T

CATACAGTATTTGGCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATCAC

MVSC

CATTCAGTACCTTGCCCACGTT-------------------------------------*** *****

* **

**

Myh11_T

GGTAGCGCTGACTCTATAAAGCCAGA--TGTCCGAAGCATACAGAGAGATTTGGACCATC

MVSC+

------GCTGATGCTACTGAGCCCCAGTCTCCTGAGGCAGAA-----------------*****

***

****

* ** *** *

Myh11_T

CCAGCCTGGGATCAGTGTCAGATCCGAGCTCTCCATCCGGTGTTCTCCTGCTAGTCCACC

MVSC+

----------------------AGCGTCGTTGCCGGAGTGTGGACTTCTCCTCTTCCACC
**

**

***

** ** **

******

Myh11_T

CCAGTAGCAGATCTGGGACCACCAAGCACAATGGCACAGAAAGGGCAGCTCAGTGATGAT

MVSC+

TCTGAGGCAGAGCTAGGGCTGGCACCCT-------------------------------* *

***** ** ** *

**

Myh11_T

CTACCCATCTACTCAGAAAAGATTGTGGACATGTACAAGGGCAAGAAGAGGCATGAGATG

MVSC+

---------------------------------------------------CAGGAGAGC
** ****

Myh11_T

CCGCCTCACATCTATGCCATTGCTGATACGGCCTACAGAAGCATGCTACAAGATCGTGAA

MVSC+

CCCCTTCATCCCTTGA-------------------------------------------** * ***

**

Myh11_T

GACCAGTCCATTCTGTGCACAGGTGAGTCTGGAGCTGGAAAGACAGAGAACACAAAGAAA

MVSC+

------------------------------------------------------------

Myh11_T

GTCATACAGTATTTGGCTGTGGTGGCATCCTCCCACAAAGGCAAGAAAGACAGCAGCATC

MVSC+

--------GCAGTCAGG------------------------------------------* * *

Myh11_T

ACGGGGGAGCTGGAAAAGCAGCTTCTACAGGCAAACCCAATCCTGGAGGCTTTCGGCAAT

MVSC+

-----------------------------------------------TCCCGCATCTTCC
*

Myh11_T

GCAAAAACTGTCAAGAATGACAACTCCTCCCGCTTTGGCAAGTTCATTCGCATCAACTTT

MVSC+

TCATACTCTGTCAAGAATGACAACTCCTCCCGCTATTACAGAAGCATGCTACAAGATCGT
** *

*************************** *

**

*** *

Myh11_T

GATGTCACTGGTTACATTGTGGGTGCCAATATCGAAACATATCTTCTGGAAAAGTCTCGG

MVSC+

GGGGGGA-AGGTAACATAGTTGGTCAA-----------AGGTCATCAGTGCAGTTTT-GC
*

*** **** ** ***

** ** *

* * *

Myh11_T

GCTATTCGACAGGCTAGGGACGAGAGAACGTTCCACATCTTCTACTATCTGATCGCTGGA

MVSC+

CCTT------CCCTTAGGGA-------CAGGGCAAGTCAGTCCAGTGTGTGGTCCTTGGA
**

******

* *

** * * * ** **

****

Myh11_T

GCCAAGGAGAAGATGAGAAATGACCTACTTTTGGAGAGCTTCAACAGCTACACATTTTTA

MVSC+

GCCAAGGGGGAAGGC--------------------------------------------******* * *

Myh10 Transcript aligned against Stem cells

Myh10T

TCACACAAAGGAAGAAAGGACCACAATATTCCTGGGGAACTTGAACGGCAGCTTTTACAA

MSCs

--------------------------------------ACTTGAACGGCAGCTTTTACAA
**********************

Myh10T

GCAAATCCAATTCTGGAATCATTTGGAAATGCGAAGACTGTGAAAAATGATAACTCATCT

MSCs

GCAAATCCAATTCTGGAATCTTTTGGAAATGCGAAGACTGTCAAGAATGACACTCCTCCC
******************** ******************** ** ***** *

Myh10T

CGCTTTGGCAAGTTTATCCGGATCAACTTTGATGTAACCGGCTATATTGTTGGGGCCAAC

MSCs

GCGATCCTCCCTCTTCTTACAGAAGCATGCTACAAGATC------------GTGAAGACC
*

** *

* *

* *

* *

Myh10T

ATTGAAACATACCTTCTGGAAAAGTCCCGTGCTGTTCGTCAAGCTAAAGATGAACGCACG

MSCs

AGTCGATT-------CTG-----------------------------------------* *

***

Myh10T

GCAAATCCAATTCTGGAATCATTTGGAAATGCGAAGACTGTGAAAAATGATAACTCATCT

MSC+

-----------------AGCTTTTGGAAATGCAAAAACTGTCAAGAATGACACTCCTCC* * *********** ** ***** ** ***** *

Myh10T

CGCTTTGGCAAGTTTATCCGGATCAACTTTGATGTAACCGGCTATATTGTTGGGGCCAAC

MSC+

CGC--TGACTGG-----------------------------------------------***

** *

Myh10T

TTCAAAAAGGAGAGGAACACTGACCAAGCTTCCATGCCCGAGAACACAGTTGCACAGAAA

MSC+

------------------------------------------------GTTA-------***

Myh10T

AAGGCAAAAAACTTAGCCAAAATCAGGAATAAGCAAGAAGTGATGATCTCGGATTTAGAA

MSC+

-------------------------------------------------------CAGAA
****

Myh10T

GAACGCTTGAAGAAGGAGGAGAAAACACGGCAGGAACTGGAAAAGGCCAAACGGAAACTG

MSC+

GCATGCTACAAGATCGTG-----------------------------------------* * ***

****

* *

Myh10T

GGTGCCGGGAAGACAGAAAATACAAAGAAAGTCATTCAGTACCTTGCCCACGTTGCTTCT

MVSCs

-----------------------------------------------CCACGTTGCTTCT
*************

Myh10T

TCACACAAAGGAAGAAAGGACCACAATATTCCTGGGGAACTTGAACGGCAGCTTTTACAA

MVSCs

TCACACAAAGGAAGAAAGGACCACAATATTCCTGGGGAACTTGAACGGCAGCTTTTACAA
************************************************************

Myh10T

GCAAATCCAATTCTGGAATCATTTGGAAATGCGAAGACTGTGAAAAATGATAACTCATCT

MVSCs

GCAAATCCAATTCTGGAATCATTTGGAAATGCGAAGACTGTCAAGAATGACACTCCTCCC
***************************************** ** ***** *

Myh10T

CGCTTTGGCAAGTTTATCCGGATCAACTTTGATGTAACCGGCTATATTGTTGGGGCCAAC

MVSCs

GCTGAGTGTTTCGATATTACAGAA------------GCATGCTACAAGATCGTGAGGACC
*

***

**** *

* * *

* *

Myh10T

ATTGAAACATACCTTCTGGAAAAGTCCCGTGCTGTTCG-TCAAGCTAAAGATGAACGCAC

MVSCs

AGTCGATTCTTTGCACGGGTG-AATCAGGTGCCGGGAAGACAGAAAATACAAAGAAAGTC
* *

* **

* **

**** *

**

* * *

Myh10T

GTTTCATATCTTTTATCAGTTGCTCTCTGGAGCAGGGGAACACCTGAAGTCCGACTTACT

MVSCs

ATTCAGTACCTTGCCCACGTT--------------------------------------**

Myh10T

** ***

***

ATCTACAACCCTGCCACTCAAGCTGACTGGACAGCTAAAAAGCTGGTGTGGATTCCATCT

MVSC+

------------------------------------------------GCTGATGCTACT
* *

**

Myh10T

GAACGCCATGGTTTTGAGGCAGCTAGTATTAAAGAAGAACGGGGCGATGAGGTTATGGTG

MVSC+

GAGCCCCAGTCTCCTGAGGCAGAAAGC--------------------------------** * ***

********

**

Myh10T

GAGCTGGCAGAGAACGGGAAGAAAGCGATGGTCAACAAAGATGACATTCAGAAGATGAAC

MVSC+

--------------------------GTCGTTGCCGGAGTGTGGA--------------*

* *

**

Myh10T

CCGCCGAAGTTCTCCAAGGTGGAGGACATGGCAGAGCTCACGTGCTTGAACGAAGCCTCT

MVSC+

-------------------------------------------------------CTTCT
* ***

Myh10T

GTTTTACACAATCTGAAGGATCGCTACTATTCAGGACTCATCTATACTTACTCGGGACTC

MVSC+

CCTCTTCCACCTCTGAGGCAGAGCTAGGG------------------------------* * *

***** * *

****

Myh10T

TTCTGTGTGGTGATAAATCCCTACAAGAACCTTCCGATTTACTCTGAGAATATTATTGAG

MVSC+

------------------------------------------------------------

Myh10T

ATGTACAGAGGGAAGAAGCGCCATGAGAAGCCGCCACACATCTACGCCATATCAGAGTCT

MVSC+

-----------CTGGCACCCTCAGGAGAGCCCCCTTC----------------------* * *

** ****

** *

Myh10T

GCCTACAGATGCATGCTTCAAGATCGTAAGGACCAGTCGATTCTTTGCACGGGTGAATCA

MVSC+

------------------------------------------------------------

Myh10T

GGTGCCGGGAAGACAGAAAATACAAAGAAAGTCATTCAGTACCTTGCCCACGTTGCTTCT

MVSC+

-----------------------------ATCCCTTGAGCAGTCAGGTCCCGCATCTTCC
*

* ** ** *

* **

****

Myh10T

GCAAATCCAATTCTGGAATCATTTGGAAATGCGAAGACTGTGAAAAATGATAACTCATCT

MVSC+

---------------------------------TACTCTGTCAAGAATGACAACTCCTCC
*

**** ** ***** ***** **

Myh10T

CGCTTTGGCAAGTTTATCCGGATCAACTTTGATGTAACCGGCTATATTGTTGGGGCCAAC

MVSC+

CGCTATTACAGAAGCATGC----------------------------------------**** *

**

** *

Myh10T

CTGGAAGGCTTTAATAACTACAGATTCCTCTCCAACGGCTACATCCCTATTCCTGGACAG

MVSC+

--------------------------------------------------TACAAGATCG
* *

**

Myh10T

CAAGACAAGGATAACTTTCAGGAGACCATGGAAGCCATGCACATAATGGGCTTCTCTCAC

MVSC+

TGGGGGGAAGGTAACATAGTTGGTCAAAGG-TCATCAGTGCAGTTTTGCCCTTCCCTTAG
*

* * **** *

* *

**

**

**** ** *

Myh10T

GAAGAGATCCTCTCAATGCTTAAAGTCGTGTCTTCAGTGCTGCAGTTTGGAAACATCTCT

MVSC+

GGACAGGGCAAGTCAGTCCAGTGTGTGG-------------------------------* * **

*** * *

** *

Myh10T

ATCAAAGTTGGCCGAGATTATGTACAGAAAGCCCAGACCAAAGAGCAGGCAGATTTTGCA

MVSC+

----------------------TCCTTGGAGCCAAGGGGG-------------------* *

**** **

Myh10T

GTGGAAGCATTGGCAAAGGCTACCTATGAGCGGTTGTTTCGCTGGCTCGTTCACCGCATC

MVSC+

------------------------------------------------------------

Myh10T

AATAAGGCGCTGGATAGGACCAAACGCCAGGGAACGTCTTTCATTGGGATCCTGGATATT

MVSC+

---------------------AAGGCCCAGGGAGTGTCACAGTATAGCAGCAGGAATAAT
**

Myh10T

*******

***

* * * *

* *** *

GATGTGGACCGAATTGTAGGTCTGGATCAAGTCACTGGCATGACTGAGACAGCTTTTGGC

MVSC+

--------------------------------------------AGTGTGAGTTT----* *

Bioinformatics results of stem cells

MSCs

MSCs+ No significant similarity found

MVSC

MVSC+

Mouse
PPC = 100.0%, Size = 240 (bp), GC content = 51.7%
Fp: Tm = 60.7 (C), G = -24.4 (kcal/mol), 3'G = -3.0 (kcal/mol)
Rp: Tm = 64.2 (C), G = -26.8 (kcal/mol), 3'G = -2.5 (kcal/mol)

** **

Binding sites: 633(24/24) ... 872(24/24)

2 Amplicons were generated
TACAGAAGCATGCTACAAGATCGTgaagaccagtccattctgtgcacaggtgagtctggagccggaaagacagag
aacac
aaagaaagtcatacagtacttggctgtggtggcatcctcccacaagggcaagaaagacagcagcatcacggggga
gctgg
aaaagcagcttctacaggcaaacccaatcctggaggctttcggcaatgcgaaaaccGTCAAGAACGACAACTCCT
CTCGC

TACAGAAGCATGCTACAAGATCGTgaagaccagtccattctgtgcacaggtgagtctggagccggaaagacagag
aacac
aaagaaagtcatacagtacttggctgtggtggcatcctcccacaagggcaagaaagacagcagcatcacggggga
gctgg
aaaagcagcttctacaggcaaacccaatcctggaggctttcggcaatgcgaaaaccGTCAAGAACGACAACTCCT
CTCGC

DISCUSSION
Where the primers bind was found, transcript was generated. The primers bind
exclusively to Myh11.

There was no significant similarity found when the primers were aligned to Myh10
transcript
Primer bond to the transcripts Myh11 when aligned to the genome.

Isolation of RNA from a whole vessel of a rat for the identification and observation of the

splice variants (Isoforms). SM1A, SM1B, SM2A and SM2B

To design a primer for these splice variant for identification and classification. This will be
done by Bioinformatics

CONCLUSION
Remind the reader about your aim
The strengths of the argument
Reflection on your research
What are the significance about your findings?
Implications for the broader filed

Findings should have implications

What are limits to your approach must match with your introduction in terms of
the ideas put forth and the arguments that are presented
Say why you couldnt conclude

Differentiated Cells
From a fresh aorta of a rat and mouse, digest the vessel into RNA, 95% of the cells are known
to be differentiated cells
Identify and characterize rat MVSCs, by verifying the existence of SM-MHC cells in the
tunica media
Generate specific RNA from tissue Fragments (~100 mg) of the muscle samples to be used
for RNA analysis by PCR
Measure and identify the splice variant in the PCR product which is SMC-1A, SMC-1B,
SMC-2A, SMC-2B
Bioinformatics: Design new primers for the variants SMC-1A, SMC-1B, SMC-2A, and SMC2B.
Amplify using specific primers
The obtained cDNAs will used as template for RT-PCR analysis. PCR reverses RT to cDNA
Run fragments (product from PCR) on gel electrophoresis to separate and compare the splice
variants.
Stem Cells

From a fresh aorta of a rat and mouse, Isolate R.N.A directly from the vessel wall
A multi potent Vascular Stem Cell population will be generated by explant and then cultured.
Explant Culture is the removal of endotherm This involves cutting the vessel out into bits,

removing the tissue with enzyme

Identification of the four splice variants of MYH11
Generation of cDNA
Design new primers
Run fragments on gel electrophoresis to separate the splice variants and compare them. The
unique difference between SMB1 and SMA is that SMB is an insert, while SMA1 has no

insert. When primers are been bind to theses variants, SMB fragment would be longer
MYH11 should not be vivid as stem cell is not a biomarker for MYH11, so no splice variant
should be vivid.

Stem Cells Inducible

From a fresh aorta of a rat and mouse, Isolate R.N.A directly from the vessel wall
A multi potent Vascular Stem Cell population will be generated by explant and then cultured
Stimulate the cells with TGF-1 to generate My11. TGF-1 is an important growth factor in
vascular smooth muscle TGF-1 (Transforming Growth Factor Beta-1) is the founding
member of the TGF super family. This family is made up of a number of different members

including Activins, Bone Morphogenic Proteins (BMPs) and growth and differentiation
factors.
Feed with JAG, JAG is a notch ligand stimuli that turns on TGF-1
My11 should now be expressed
Analyse the different splice variant illustrated by stimulation
Run fragments on gel electrophoresis to separate and compare the splice variants.

ACKNOWLEDGEMENTS
REFERENCES
1. Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schffer, Jinghui Zhang,
Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSIBLAST: a new generation of protein database search programs", Nucleic Acids Res.
25:3389-3402.
2. Mooney, C. J., Hakimjavadi, R., Fitzpatrick, E., Kennedy, E., Walls, D., Morrow, D.,
et al. (2015). Hedgehog and Resident Vascular Stem Cell Fate. Stem Cells
International, 2015(6), 46842816

3.