Its a complicated question. First I wanted to ask if you wanted to work on gene level, why to go a hard way of amplifying the cDNA. What is your purpose for that? just curious!

Now coming to the question, it could be possible that your RNA was contaminated with DNA. Also, there might be possibility as you said there was no amplification, but in that case you only see primer dimers rather multiple bands. It is generally seen that there is not absolute conversion of RNA to cDNA. Therefore, you could have RNA and cDNA together in the reaction which gave multiple bands. Another reason could also be the splice variants.

As a confirmation of your cDNA, may be you can do a PCR with mRNA primers, or you can go for cheap sanger sequencing. Sequencing would serve two purpose. It will confirm whats in the PCR product and what all splice variants are present, if any. Or do sort of gradient-time PCR to make sure all the RNA present in reaction is converted to cDNA.

1. Why do want to use cDNA to amplify a gene- usually we check transcripts? What is your experiment?

2. If you are getting the correct genomic fragment- why use cDNA?

3 Are your primers across intron-exon junctions?.

4. Are you trying to show intron retention in your transcript? Then your cDNA may give- a)hetero nuclear RNA amplification which will be the same size as your genomic band suggesting that the splicing has not occurred, b) another smaller band (minus the size of the intron) could be the spliced transcript. If this is true then the product sizes should add up to one or the other. As Abhijeet suggests- sequence the PCR product, may have primer dimers, DNA contamination. What controls are present in your PCR reaction- -RT control, non-template control, positive control?

are you running a control like actin to check that there is cDNA present and that the transcription and pcr are working?

Its a confounded inquiry. In the first place I needed to inquire as to whether you needed to deal with quality level, for what reason to go a hard method for opening up the cDNA. What is your motivation for that? simply inquisitive!

Presently going to the inquiry, it could be conceivable that your RNA was defiled with DNA. Likewise, there may be probability as you said there was no intensification, yet all things considered you just observe preliminary dimers rather different groups. It is for the most part observed that there isn't outright change of RNA to cDNA. In this way, you could have RNA and cDNA together in the response which gave numerous groups. Another reason could likewise be the graft variations.

As an affirmation of your cDNA, might be you can complete a PCR with mRNA groundworks, or you can go for shoddy sanger sequencing. Sequencing would fill two need. It will affirm whats in the PCR item and what all join variations are available, assuming any. Or then again do kind of inclination time PCR to ensure all the RNA exhibit in response is changed over to cDNA.

regards

This guy @Hassan Nima is soo shameless and stupid fraud. I wonder who gave him a PhD degree and made him a lecturer. I feel pity for the unfortunate institute, that he belongs.

I am sorry for not seeing so many answers on my post. So I will start with my query and answers one at a time. Thank you all for answering.

Abhijeet Singh @Abhijeet Singh want to amplify it via cDNA as my gene contains intron and use it for cloning experiments. I have made sure that there is no DNA contamination by purifying my RNA after RNA isolation, also made sure by loading RNA on to an agrose gel and checked its integrity and purity.

Before starting up with my experiments I made sure that my gene doesnt have any splice variants. So that ambiguity of alternative variants gets cancelled.

I always put a control with my PCR reactions. So there was a positive control with my reaction. Positive control was gDNA+ gene primers for which I always get result. Experimental- cDNA + Gene of interest and gene of interest + genomic DNA (using gene specific primers). Gene is amplified along with the intron using gDNA but in cDNA multiple bands.

I am confused with my results plus this is the first time I made a cDNA.

Paul Rutland@Paul Rutland I did not think of using actin primers along with cDNA. I will surely do that once. As I was having less amount of cDNA, I tried amplification with only my gene of interest. All the PCR components are working fine as I put a positive control with my reactions. I am getting a band of expected size in positive control. No bands in negative control.

If amplifying a housekeeping gene ( borrow primers from another researcher) works then you have cDNA so the problem is just the multiple bands. Some common ways of making pcr more specific are running a temperature gradient from 4c above to 5c below the primer Tm as Abhijeet Singh says. Alternatively run the pcr reaction in the presence of up to 8%DMSO in steps of 2% DMSO . If you have room to design nested pcr primers ( or even half nested with just one new internal primer ) you can re amplify your dirty product and often you get just the correct band with the use of the second set of primers. Finally touchdown pcr works well when one primer is more specific than the other primer as it enriches the template before the proper pcr with both primers starts to work

Paul Rutland @paul I have already added DMSO in my PCR components as the gene is GC rich. I will try rest of the suggestions given by you.

Thank you so much.