RT-PCR Protocol:

Materials Needed:

 SuperScript® VILO™ cDNA Synthesis Kit – Sold by Invitrogen by Life Technologies – Catalog
number 11754-050 – $472.00
 iTaq™ Universal Probes Supermix – Sold by Bio-Rad – Catalog number 172-5130 - $123.00
o 2x qPCR mix, contains dNTPs, iTaq DNA polymerase, MgCl 2, enhancers, stabilizers, and
ROX normalization dyes
 Control primer/probe set (i.e. GAPDH or HPRT – or appropriate control genes for target tissue)
 Target primer/probe set (both can be bought through IDT)
 AllPrep DNA/RNA Mini Kit (50) – Sold by Qiagen – Catalog number 80204 - $459.00
 RNaseZap RNase Decontamination Solution – Sold by Life technologies – Catalog number
AM9780 - $63.50
 Hard-Shell® Thin-Wall 384-Well Skirted PCR Plates – Sold by Bio-Rad – Catalog number HSP-3805 -
$317.00
 PrimeTime Predesigned qPCR primer and probe – IDT – $ Variable

Procedure:

1. RNA extraction from frozen tissue

a. Follow Qiagen protocol for simultaneous purification of Genomic DNA & Total RNA
b. Be careful not to work in a dirty environment. RNase contamination may degrade RNA,
use RNaseZap thoroughly.
c. Optional: Use Qiagen’s supplementary protocol - Acetone precipitation of protein from
Buffer RLT or Buffer RLT Plus lysates
d. RNA will be used for sensitive downstream application, so perform optional DNase
digestion using steps E1-E4 in Qiagen DNA/RNA protocol.
e. Determine concentrations of RNA for use in cDNA synthesis by Nanodrop.
2. cDNA synthesis using Superscript Vilo cDNA synthesis kit
a. For a single reaction, combine the following components in a tube on ice. For multiple
reactions, prepare a master mix without RNA.

Reaction Component x 1 sample (µl) x # samples

5X VILO Reaction Mix 4
10X Superscript Enzyme Mix 2
RNA (up to 2.5 µg) 250 ng/µl
DEPC-treated water to 20 µl
b. Gently mix tube contents and incube at 25°C for 10 minutes.
c. Incubate tube at 42°C for 60 minutes.
d. Terminate reaction at 85°C for 5 minutes.
 e. Steps b-d can be done in thermal cycler with preset program and left overnight on 4 or
12°C hold. Use diluted or undiluted cDNA in qPCR or store at -20°C until use.
3. RT-PCR using PrimeTime Predesigned qPCR assays
a. If a 384-well plate is being used, 10 µl reactions may be used.
b. Samples should be run in replicates, either triplicate or quadruplicate.
c. A control gene should be used for normalization. A list of control genes is provided on
page 41 of IDT’s qPCR Application Guide.
d. Resuspension of PrimeTime qPCR Assay
i. Centrifuge PrimeTime qPCR Assay tubes at 750 x g for 10 sec prior to opening in case
any material was dislodged during shipment
ii. Resuspend assay in IDTE (10 mM Tris, 0.1 mM EDTA, pH 8.0) at the volumes indicated in
table.
1. Resuspend PrimeTime qPCR Assays as 40X, 20X, or 10X stocks. A particular
concentration may be preferred depending on the size of the assay and desired
final reaction volume. (20X is generally used).
2. Vortex the sample to ensure maximal product recovery.

iii. Centrifuge the resuspended assay at 750 x g for 10 sec. The resuspended assays will
yield a final 1X concentration of 500 nM primers and 250 nM probe when ordered using
default conditions.

e. Prepare a Master Mix containing iTaq Universal Probes Supermix (2X), each Primer-
Probe (control gene and target gene), and PCR-Grade water. An example of a 10µl
reaction with 20X resuspended primers is shown below:

Component Vol. Per 10 µl Reaction X 96 samples

iTaq Universal Probes Supermix 5.0 µl 480 µl
Control Gene (HPRT) 0.5 µl 48 µl
Target Gene (Slc6a4) 0.5 µl 48 µl
PCR-Grade Water 3.0 µl 288 µl
 f. Upon setting up the Master Mix for each target gene, pipette 9 µl of Master Mix into
each designated well on a 384-well plate.
g. After Master Mix has been added to each well, add 1 µl of each cDNA sample into its
respective well. Mix contents of each well by pipetting up and down upon adding cDNA.
h. Use clear plate-sealing film and cover the entire plate. Centrifuge for 2 minutes at low
speed (800-1000g) to clear wells of bubbles and bring contents to bottom of well.
i. Place 384-well plate in CFX384 Real-Time System.
4. RT-PCR Protocol
a. Run in Bio-Rad CFX384 Real-Time System using CFX Manager software.
1. Polymerase activation & DNA denaturation – 95 °C for 0:30 seconds
2. Denaturation – 95 °C for 0:05 seconds
3. Annealing/Extension – 60 °C for 0:30 seconds
a. + Plate read
4. Repeat steps 2 – 3 (denaturation & annealing/extension) 54 times.