Hi, I am using VASA-seq for RNA sequencing. The last two steps of the protocol are cDNA synthesis (via reverse transcription) and PCR. I had been using Superscript III for cDNA synthesis and was getting a lower PCR yield. Then I switched to Maxima H Minus Reverse Transcriptase. The PCR yield increased dramatically, but I am getting this weird around 1200 bp long fragments (see the attached figure). My expected peak is around 300 bp. I have attached a figure of the fragment size distribution of the PCR DNA (analyzed on fragment analyzer).
#fragment_analyzer #PCR #VASA_seq #Maxima H Minus Reverse Transcriptase #SuperscriptIII
It's difficult to interpret this result because you don't have a baseline - the red line on this graph should be horizontal and flat. The most likely cause is that the high-MW contaminating material is a broad smear from ~400nt to well over 3000nt, so your upper marker is superimposed on top of it, and the high-MW peak is still passing the sensor when the measurement cuts off (right edge of the graph).
It would help a lot to see some images of the results you were getting when using Superscript III. My gut feeling is that the increase in PCR yield you're seeing is almost entirely junk.
This info would also help a lot:
1. Have you measured the cDNA concentration? Is there a difference between SSIII and Maxima?
2. How many PCR cycles are you doing, and how did you choose this number?
3. Does your library/adapter include UMI or other means of removing PCR duplicates from your data?
Dear Ms. Leighton,
I am terribly sorry for taking too long to get back to you. I confess that this is not very professional on my part.
Fortunately, the problem is fixed. I realized that I was using the wrong thermocycler program for Maxima. I was using 50°C for 60 min for the enzyme activity and then 70˚C for 15 min to deactivate the enzyme.
The correct program is 50°C for 30 min for the enzyme activity and then 85˚C for 5 min to deactivate the enzyme. This program worked and gave a nice fragment size distribution.
I also tried SuperScript IV at 50°C for 10 min for the enzyme activity and then 80˚C for 10 min to deactivate the enzyme.
I had even better results with SuperScript IV and decided to use it for this step from now on.
Nevertheless, here are the answers to your questions:
1. I measured the cDNA concentration, which indeed was higher with Maxima.
2. I was doing 9 PCR cycles, which I didn't calculate. I was simply following the established protocol.
3. Yes, we use UMI to remove PCR duplicates.
Some extra info: I also considered the possibility of PCR bubbles. So, I did PCR with higher primer concentration, but it didn't help.
Many thanks for your response and suggestions, Ms. Leighton.