Effect of a Shortened Reverse Transcription Time on qPCR Amplification

Reverse transcription (RT) is a fundamental and widely used reaction in molecular biology research. It is a critical reaction for many basic techniques and applications such as qPCR, endpoint PCR and cDNA cloning among others. The transcription of an RNA template to a new strand of cDNA occurs in 15–80 minutes, depending on the procedure used. Three types of primers can be used for this reaction: Random primers, oligo(dT) primers and gene-specific primers. The type of primer selected will depend on the RNA template and downstream application.

GoScript™ Reverse Transcription Mixes are convenient reagents that include an optimized set of components designed for efficient first-strand cDNA synthesis. Priming using random primers is the most general method of initiating cDNA synthesis from a variety of RNA templates. While random primers can be used to prime first-strand cDNA synthesis from all RNA molecules, oligo(dT) priming initiates first-strand synthesis by annealing to the 3´ end of any polyadenylated RNA molecule. Therefore, oligo(dT) is widely used to preferentially reverse transcribe messenger RNAs (mRNAs). GoScript™ Reverse Transcription Mixes are supplied with either Oligo(dT) or Random Primers. The protocol for the GoScript™ Transcription Mixes recommends a cycling protocol of 80 minutes for RT. In the following experiments, we tested whether the time required for the RT reaction could be reduced. We tested 30- and 15-minute shortened protocols compared to the original 80-minute recommended protocol. Three different transcripts, GAPDH, SDHA and UBC, with high, medium and low expression levels, were tested using dye-based qPCR and cDNA generated with these shortened protocols. Results were analyzed and performance was compared based on efficiency and linearity (r2).

Materials

• Universal Human Reference RNA (Agilent, Cat.# 740000; 1µg/µl)
• Nuclease-Free Water (Cat.# P1195)
• GoScript™ Reverse Transcription Mix, Random Primers (Cat.# A2801)
• GoScript™ Reverse Transcription Mix, Oligo(dT) (Cat.# A2791)
• GeneAmp® PCR System 9700 (Applied Biosystems)
• GoTaq® qPCR Master Mix (Cat.# A6002)
• 7500 Real-Time PCR System (Applied Biosystems)
• Primers (1) to amplify targets of three different sizes as shown in table below

Protocol

An eight-point standard curve of Universal Human Reference RNA was made in Nuclease-Free Water with tenfold dilutions of 200ng/µl–0.02pg/µl.

Reverse transcription was performed using the GoScript™ Reverse Transcription Mix Random Primers and GoScript™ Reverse Transcription Mix Oligo(dT) in triplicate with 5μl of RNA (1μg–0.1pg of total RNA) in a final volume of 20µl. With the highly expressed GAPDH target, 0.1µg–0.1pg of RNA was used as input. For SDHA and UBC, 1µg–1pg was used as input. Reactions were assembled according to the GoScript™ Reverse Transcription Mix protocols [#9PIA280 for random primers and #9PIA279 for oligo(dT)].

Three reaction times were tested, the 80 minute standard protocol plus two shortened reaction times of 30 and 15 minutes. Shortening the enzyme inactivation step to 5 minutes at 95°C is an option mentioned in the protocols for the GoScript™ Reverse Transcription Mixes #9PIA280 and #9PIA279.

Reverse transcription conditions tested are shown in the table below.

Real-time qPCR was performed with 2µl of reverse transcribed cDNA in a 20µl total reaction volume using GoTaq® qPCR Master Mix using the cycling conditions recommended in the GoTaq® qPCR Master Mix Technical Manual #TM318 to amplify each target (GAPDH, SDHA and UBC).

Reverse transcription was performed with serially diluted RNA using GoScript™ Reverse Transcription Mix with Random Primers or Oligo(dT) and 3 different reactions times: 80 minutes, 30 minutes and 15 minutes. Following reverse transcription, qPCR was performed with 2µl of RT products to analyze gene expression.

Figure 1 shows the Cq values obtained for GAPDH with both primer mixes. Similar Cq values were obtained with all three reverse transcription reaction times. Tables 1 and 2 summarize percent qPCR efficiency and linearity (r2) for each target and reaction time. Ideal efficiency is 100% with a linearity (r2) of 1, but an efficiency in the range of 90–110% and a linearity (r2) ≥0.98 is acceptable according to the MIQE guideline. (2)

With GoScript™ Reverse Transcription Mix, Random Primers, similar performance was obtained with all reaction times tested for each target transcript. With GoScript™ Reverse Transcription Mix, Oligo(dT), similar performance was obtained with the three reaction times for GAPDH, whereas efficiency and linearity are out of the range for SDHA and UBC during the 15 minute reaction. Fifteen minutes was sufficient to produce the 780bp cDNA needed to amplify GAPDH, but not enough to amplify the SDHA and UBC cDNAs of 2,301bp and 2,251bp, respectively.

We found that reverse transcription with GoScript™ Reverse Transcription Mix, Random Primers, can be reduced to 30 or even 15 minutes without an effect on qPCR performance for the transcript sizes tested (780bp–2,301bp). When using GoScript™ Reverse Transcription Mix, Oligo(dT), reaction time could be reduced to 30 minutes without affecting qPCR performance for the three transcripts tested. However, shortening the RT reaction using oligo(dT) to 15 minutes only amplified GAPDH at 780bp, but the other transcripts over 2kb were negatively affected, suggesting that the full-length cDNA was not synthesized.

When designing assays with GoScript™ Reverse Transcription Mixes, shortened reaction times should be tested and compared with the standard 80-minute protocol to ensure acceptable reverse transcription is achieved.