Good quality starting material is king for reverse transcription! Obtaining reliable results in any experiment requires good preparation. We often take reverse transcription for granted, and we don’t always consider that our qPCR might be performing poorly because of problems in that step. Since it’s quite often the reverse transcription reaction itself that causes fuss in qPCR, we at Bitesize Bio have collected some common pitfalls that can arise when performing this technique and offer you some advice for overcoming them.
Let us proceed!
Pitfall 1: The RNA Has Disintegrated!
When electrophoresing RNA for quality assessment, degraded RNA appears as a smear in agarose gels, rather than sporting the characteristic ribosomal RNA (rRNA) bands (e.g., 28S and 18S subunits in mammalian RNA).
Solution: Bear These 3 Points in Mind
- Aseptic technique should be used when isolating and pipetting RNA solutions—clean the bench with an RNase inactivating solution (bleach or hydrogen peroxide also work here), use a separate set of pipettes for RNA isolation and use only nuclease-free water (make your own by treating distilled water with diethylpyrocarbonate (DEPC)).
- If you are isolating RNA from a tissue that requires storage (because, for example, your samples come from a medical center that is miles away from your lab), place the samples in liquid nitrogen or in an RNA-preserving buffer. The latter is a magic potion that permeates your tissue and keeps the RNAses inactive.
- Use an RNase inhibiting agent during reverse transcription to tame any RNAses present during the reaction (most commercial kits contain an RNAse inhibitor).
- Captivate a student that will measure the RNA concentration immediately after isolation while you prepare the reaction mixture. Be sure to keep the rest of the sample on ice. Aim to reverse transcribe your RNA as soon as you’ve worked out the concentration! This way you avoid putting the precious, fragile RNA material on hold in the -70°C freezer, and instead you can store its more robust twin brother—cDNA.
Pitfall 2: Intrusive gDNA Gets Co-Isolated with RNA
More often than not, you will encounter genomic DNA (gDNA) in you RNA sample, and this can dramatically bias your subsequent experiments.
How do you know if you have gDNA? After RT, perform a quick PCR with primers designed to span an intron-exon boundary. If you get two bands, one larger that the other, genomic DNA is there and in quite a large amount!
Another way is to make a control reverse transcription reaction where the reverse transcriptase is omitted. If you get an amplicon from this sample, it is probably gDNA contamination.
Genomic DNA (if your amplions of interest span exon-intron boundaries) can also be spotted as an additional peak in melt curve analysis when performing a qPCR with fluorescent dye chemistry.
Solution: There Are Two Time Points at Which to Approach This Issue
- Before it happens! Be extremely careful in the isolation step. With acid phenol-choloroform extraction, do not pipet the entire aqueous phase (less yield is better than more bias) so that you do not accidentally pick up some organic phase and transfer gDNA. In contrast to acid phenol-chloroform extraction, protocols involving column purification of RNA often include a DNAse treatment step during RNA isolation and, thereby, minimize the likelihood of DNA contamination ruining your perfect experiment!
- After the unlucky event! Resort to DNA removal kits that will degrade the intruder. This requires extra effort (time and cost!) to inactivate and remove the DNAse, but many people swear these kits are worth the hassle. Bearing in mind that tough times create strong personalities, this additional step will only make you stronger!
Pitfall 3: You Primed the RNA with Oligo-dT but Got No Product in Your qPCR Experiment
While oligo-dTs are very good for selectively transcribing messenger RNA (mRNA), they do exhibit some 5’ bias given the fact that they bind to poly-A tails at the 3’ ends of mRNA. What does that mean? It means that the reverse transcriptase may not have enough time to reverse-transcribe the entire mRNA into cDNA, resulting in untranscribed 5’ tails. If your gene-specific primers are designed to anneal to the 5’ end, they may not have the sequence to which to stick, and you risk a false-negative result.
Also, check your enzyme specifications to see the maximum length of cDNA fragments that can be produced (most of them go for up to 12kb, but there are some newer ones that can reverse transcribe transcripts of up to 20kb and beyond!).
Another possible reason for no amplification after oligo-dT priming is a low quality (fragmented) template or RNA with significant secondary structure.
- The first go-to solution is kind of a no-brainer—redesign your PCR primers to anneal near the 3’ end.
- If new primers don’t work, then it might be time to dig out the random hexamers*! These bind to different locations on all RNA species, transcribing everything so that the location for primer annealing will not be so critical.
- Remember to ensure that your target actually has a poly-A tail before you start reverse-transcription! Viral or histone RNAs don’t have poly-A tails so oligo-dTs are never going to work here!
However, we all know that very little in life comes without a price, and life science is no exception! Using random hexamers may lead us to another issue:
Pitfall 4: You Get No Product in QPCR after Priming with Random Hexamers
Being non-specific, random hexamers will stick to any RNA species; therefore, the majority of resulting cDNA will be of rRNA origin, since rRNA is the most abundant RNA species. How does this affect your experiment? If the gene of interest is weakly expressed, the amount transcribed into cDNA may not be enough for you to detect it in qPCR.
- Go back to oligo-dTs as their high specificity should overcome this problem.
- If you do not plan on using the RNA for detecting other genes, resort to gene-specific priming with your gene-specific primers and carry out one-step RT PCR. Nothing can be more specific than that.
*Note: Bear in mind that priming with random hexamers can overestimate mRNA copy number by up to 19-fold. To control for this while doing your gene-expression analysis, you should ensure that the calibrator samples are transcribed using the same method of priming. In other words, if you are going to deliberately make an error, do it uniformly across the entire experiment!
In the end, as we all know, every optimization step carries a trade-off—you win some, you lose some. If components that suit your experiment are not available, then make sure you perform a well-thought-out experiment, which is executed with precision and properly analyzed. This will always be better than a super-specific experiment performed in haste, with low precision and incorrect controls.