Why couldn't I find the polyA tail sequence in the 3' RACE PCR sequencing results?

04 May 2023 1 7K Report

Recently, I did the 3' RACE PCR (Thermofisher 3'-RACE PCR kit) using the total RNA and sent it for sequence (I used a gene-specific primer with AUAP). But in the sequence result, we couldn't find the polyA tail. What would be the reason for that? (I have checked the 3´ RACE system, and there is no problem in the system.)

Nikolay Klyashtornyy

There are several reasons why the polyA tail sequence may not be detected in 3' RACE PCR sequencing results. Here are a few possibilities:

Low quality sequencing: The polyA tail sequence may be present in the sequencing data but may not be detected due to low sequencing quality. In this case, re-sequencing the sample with a higher sequencing depth or using a different sequencing platform may be helpful.

Degraded RNA: If the RNA used for the 3' RACE PCR is degraded, the polyA tail sequence may not be present. RNA degradation can be caused by improper handling, storage or isolation of RNA. To prevent RNA degradation, it is important to use RNase-free reagents and to store RNA samples properly.

Primer design: The gene-specific primer used in the 3' RACE PCR may not have been designed properly. It is important to design a primer that anneals to the gene of interest and is long enough to avoid non-specific amplification. Additionally, the primer may need to be positioned further downstream to ensure amplification of the polyA tail sequence.

RNA modification: Some RNA modifications, such as m6A, can interfere with the annealing of the oligo(dT) primer used in the reverse transcription step. This can result in incomplete cDNA synthesis and may explain why the polyA tail sequence is not detected. In this case, using a modified oligo(dT) primer or performing reverse transcription with random primers may be helpful.

PolyA tail length: The length of the polyA tail can vary between RNA molecules and can affect the efficiency of the 3' RACE PCR. In some cases, the polyA tail may be too short to be detected by PCR. In this case, using a polyA tail extension kit or designing a different gene-specific primer closer to the polyA tail may be necessary.

It is important to troubleshoot the experimental design and technique to determine the cause of the missing polyA tail sequence in the 3' RACE PCR sequencing results.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

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