cDNA synthesis using Superscript IV and general qPCR

Published

November 12, 2019

cDNA synthesis

Background

RNA abundance-analysis is enabled through the synthesis of complementary DNA from single stranded RNA prior to real-time quantitative polymerase chain reaction (qPCR). Successful cDNA synthesis is made using high quality input RNA. This protocol is slightly modified from the Superscript manual.

Materials

  • Heating block or thermal-cycler capable of 65, 50-55 and 80°C
  • RNase-free tubes, pipette tips
  • Superscript IV kit (SSIV Buffer, SSIV Reverese transcriptase, 100 nM DTT)
  • Oligo dT and/or Random Hexamer primers
  • 10 mM dNTP mix
  • Total-RNA or mRNA

Methods

  1. Combine the following components per reaction (consider creating a master mix from component a and b if you are running many samples, master mix 1):
Component Volume per reaction
50 μM Oligo dT or 50 μM Random hexamers or 25 μM Oligo dT and 25 μM Random Hexamers 0.5 μl Oligo dT (art nr. AB-1247) + 0.5 μl Random Hexamers (SO142)
10 mM dNTP mix (10 mM each) 1 μl
Template RNA (0.1-5000 ng total-RNA) Up to 11 μl
Nuclease-free water Add to total volume 13 μl
  1. Mix all components, vortex and briefly spin down.
  2. Heat the mix 5 min at 65°C followed by at least 1 min incubation on ice.
  3. Bring the 5x SSIV buffer to room-temp. Vortex and spin briefly.
  4. Combine the following into master-mix 2:
Component Volume per reaction
5x SSIV Buffer 4 μl
100 mM DTT 1 μl
RNase OUT 1 μl
Super Script reverse transcriptase 1 μl
  1. Vortex and briefly spin the components of master mix 2, add master-mix 2 to the reaction tube.
  2. Incubate the reaction tube at 23°C 10 min.
  3. Incubate the reaction mix at 50-55 °C 10 min followed by 10 min at 80°C
  4. Dilute the cDNA to desired concentration (e.g. 1:25)

qPCR

Background

Targeted amplification of cDNA using specific primers is a sensitive technique for the evaluation of gene-abundance.

Materials

  • A real-time PCR machine (We use QuantStudio 5)
  • A qPCR reaction plate
  • Nuclease-free water and pipette tips
  • SYBR-green Master mix

Methods

  1. Combine a master-mix:
Component Volume per reaction
Sybr-green 2X master-mix 5 μl1
Primermix, Forward and Reverese 5 μM2 each 1 μl
H2O 2 μl
  1. Load the plate with primer-specific master-mix
  2. Add 2 μl cDNA sample.

Footnotes

  1. The final volume of the reaction may be optimized, 10 ul is a good starting point↩︎

  2. Primer concentrations might need further optimization↩︎