Neslihan Sönmez 2073542 Total RNA Isolation Report

1.Lysis step include lysis buffer and inside of the buffer there is chemicals like EDTA,SDS
and sodium chloride. The isolation process begins with the destruction of the material from
which the RNA will be isolated with an organic solvent. Guanidine isothiocinate and sodium
such as dodecyl sulfate (Sodium Dodecyl Sulfate; SDS) chemicals are widely used for the
destruction of the material used for RNA isolation and for the inhibition of RNase activity
(Nilsen, 2013). iBuffers stabilize the pH when the cells are separated. Tris-HCL stands as one
of the most common chemicals for buffering at pH 8. Sodium chloride salt can also increase
the ionic strength, increase the total concentration of solutes outside the cells. This last point
has some advantages because water can spread throughout the cell membrane from regions
with a low solute concentration to regions with a high solute concentration. Detergents
dissolve the cell membrans, allowing the cell contents to escape.Sodium chloride is used to
stabilize the lysis buffer. ii

Wash buffer 1 contains chaotropic agents that enhance in RNA adhesion to the silica
membrane and inhibits RNase activity. Wash buffer 1 and 2 have ethanol inside. In ethanol
precipitation, positively charged salt molecules (sodium acetate, ammonium acetate, lithium
chloride) are used to reduce the hydrophilic structure of RNA.iii The important aim of utilizing
multiple washing buffers in a certain order is to prevent nucleic acid eliminations and promote
purity by lowering ionic strength. As a result, we use two types of buffers that differ in terms
of ionic strength and the amount of ethanol they contain.

Positively charged salt molecules neutralize the negative charge of RNA. Ethanol, on the
other hand, allows salt molecules and RNA to interact more easily, allowing RNA to be
precipitated comfortably after centrifugation.Adding salts help the precipitation. iv

Elution step include elution buffer that contains Tris. It permits RNA to disconnect from the
matrix in the solution by disrupting the connections between RNA and DNA.

2.Spectrophotometric measurements rely on passing light through the prepared solution in

specific spectra and calculating how much of that light is absorbed by the solution. DNA and
RNA solutions absorb at 260 nm, while protein solutions absorb at 280 nm. The purity of
nucleic acids is determined by the ratio between 260 and 280 nm (A260/A280). The
A260/A280 ratio is an indication of the level of protein contamination in the sample. Pure
RNA has an A260/A280 ratio of 2.1, but values between 1.8-2.0 are considered acceptable for
most protocols.For our group(sec1-g1),the result was 2.096 which is optimal ratio for our
experiment.

Abnormal 260/230 results could suggest a problem with the sample or the extraction
technique, thus both should be taken into account. Carbohydrate transport could cause a
low A260/A230 ratio (usually a problem with plants). Phenol residue from nucleic acid
extraction is another cause of low ratio. A low 260/230 ratio indicates excessive salt
contamination, particularly guanidium salts in the Lysis buffer, which protect nucleic acid
from nucleases. A ratio of ~2.0 is generally considered "pure" for RNA.Our result was
1.127. The reason for the low rate may be indicative of the pollutants absorbed at 230 nm.
A good 260/280 ratio indicates that our sample is devoid of protein impurities, but our
260/230 is low, indicating that nucleotide contamination exist. The most likely cause for
this is that our sample has degraded.v

Because the Uracil ratio of RNA is larger than that of thymine, it has a higher 260/280
ratio. This ratio is utilized as a secondary nucleic acid purity indicator. For "pure" nucleic
acid, the 260/230 values are often greater than the 260/280 values.

3. We used extraction methods for Solid-Phase DNA

McCormick originally described a solid-phase DNA extraction method using blood

samples in 1989. This approach made use of silica-based particles, which interacted with
proteins and allowed for DNA purification. Since then, other techniques for solid/liquid
DNA extractions have been developed, with the majority of these being transformed into
commercially accessible extraction kits. To assure cell breakdown, several of the
treatments follow similar procedures. Following that, DNA adsorption, adsorbed DNA
washing, and the most recent elution processes are carried out.vi

Only the upper liquid phase containing RNA should be removed during isolation to avoid
contamination and contact of the pipette with other phases should be. After the phase is
removed, the liquid is treated with isopropanol to improve the purity of the extracted
RNA.vii

RT-qPCR method can help to obtain contamination. DNA contamination will lead to non-
specific amplifications and overestimate of transcript level. For example, if DNA
contamination occurred during the RNA separation process, we might end up with tainted
DNA for RT-PCR. We acquired tainted DNA from the extracted RNA process, therefore
 this DNA is from that as well. As a result, there are two different methods to get tainted
DNA. minus-RT control can detect contamination and set differences for each route
during the PCR procedure.

4.RNA is hydrophilic and it is easily dissolved (Rio et al., 2010b). Therefore, reducing the
hydrophilic nature of RNA. In addition, it is recommended to precipitate samples with ethanol
to increase RNA purity. RNase must be isolated quickly due to its degrading effect.viii

These enzymes are very problematic during RNA isolation because they are ubiquitous and
very difficult to eradicate. When isolating total RNA, you need to be sure to eliminate
RNAase. RNA isolation is very difficult. It is not as stable a molecule as DNA. Degradation
of RNAs during isolation is very common. The ribose sugar in RNA is one of the main causes
for its instability and sensitivity. The ribose sugar in RNA makes it more reactive than the
deoxyribose sugar in DNA. In alkaline settings, the ribose sugar causes the RNA to become
unstable. The structure of the RNA is another cause. For example, RNA contains larger
helical grooves than DNA, making it more appropriate for enzyme exposure.

The RNase enzyme can continue its activity even after autoclave, as it does not need a metal
ion cofactor. Therefore, when using the reagent, it should be noted that there is no RNase in
it.ix
i
Babenka, A. S. (2019, September 18). What is the difference between the 2 washing buffers in
silica-based DNA extraction? Retrieved from https://www.researchgate.net/post/What-is-the-
difference-between-the-2-washing-buffers-in-silica-based-DNA-extraction

ii
Hurst, T. et al (2018, May 10). Quantitative Understanding of SHAPE Mechanism from RNA
Structure and Dynamics Analysis. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311105/

iii
Laurell, H. et al (2012, January 6). Correction of RT–qPCR data for genomic DNA-derived
signals with ValidPrime. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326333/

iv
Shehadul Islam, M. et al (2017, March 8). A Review on Macroscale and Microscale Cell Lysis
Methods. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6190294/

v
Paska, C. et al (2019, March 28). Elimination of bacterial DNA during RNA isolation from
sputum: bashing bead vortexing is preferable over prolonged DNase treatment. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438495/

vi
William, V. W. et al. (1992). Effect of RNA Concentration on cDNA Synthesis for DNA
Amplification. Retrieved from https://genome.cshlp.org/content/2/1/86.full.pdf

vii
Sambrook J, Russel D. Molecular Cloning: A Laboratory Manual. Third Edition. New York,
USA: Cold Spring Harbor Laboratory Press, 2001; p. 613.
viii
Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Ethanol precipitation of RNA and the use of carriers.
Cold Spring Harb Protoc 2010b; 6: 1-5.
ix
Sokurenko, Y., Ulyanova, V., Zelenikhin, P., Kolpakov, A., Blokhin, D., Müller, D., Klochkov,
V., & Ilinskaya, O. (2016). The Role of Metals in the Reaction Catalyzed by Metal-Ion-Independent
Bacillary RNase. Bioinorganic chemistry and applications, 2016, 4121960.
https://doi.org/10.1155/2016/4121960