I have been trying to amplify a sequence of 2.2Kb by reverse transcription PCR from the HepG2 cell line. The band size I get is below 2Kb (close to 1.5Kb). I performed touchdown PCR and I added 2% Betaine in RT and also used 5% DMSO in PCR. Could it be that this cancer cell line has a large deletion? Or could there be secondary structures in the DNA template? Or what other reasons could there be?
Hi Sophia,
you have several options to find out if there is a deletion in your HepG2 sequence:
- get the amplified fragment sequenced
- use internal primers to identify possible deleted regions
- use DNA from another cell line as a template for amplification (maybe one reported to be useful as a positive control)
Best,
Christian
Sophia,
You said you amplified by reverse transcription PCR. I therefore suppose that you amplified RNA and that the forward and reverse primers are located on different exons. Did you looked at the alternative transcripts synthesized from your gene of interest (available at ensembl.org)?
Hi,
You should ensure that the reverse transcriptase you used for cDNA synthesis has a high processivity or capability to extend upto 2.5 kb. That could be the issue here.
Philippe Fort, thank you. Do you think you can please elaborate about how I find where the variant are, and which regions are different?
Go to https://www.ensembl.org and go to the Human genome, type the name of your gene. Once on the gene page, click on "show transcripts table" in the main window. You'll get the sizes of all alternative transcripts. The differences between transcripts are figured on the graph below the table.
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