I am trying to isolate a gene from rice whose size is 1.4 Kb. I have synthesized cDNA. Then In PCR of C-DNA I am getting amplification in House keeping gene (actin) but in case of my gene of interest I am getting PCR band of 3 kb. What will be the possibles reasons of getting non specific amplification. should I need to try the PCR of genomic DNA also.
Please guide me.
Thank you !
Dear Harshraj
Did you removed genomic DNA contaminations from RNA samples prior to cDNA synthesis. If not then, the amplification is from genomic DNA and might have know that it amplifying along with introns and hence the PCR product is 3 kb instead of 1.4 kb. And another possible reason might be that cDNA is not intact, it is sheared, I think so.
If you want to amplify the gene strictly from cDNA then ensure the integrity and quality of isolated RNA then treat this RNA with DNase I according to the protocol and then you can start to cDNA synthesis.
And As s suggestion, use a good Taq pol, You can use Platinum master mix from Invirogen, Hope you will get the result.
All the best
Chet RAm
Thank you Dr. Ram for your response.
I guess RNA I isolated is of good quality its almost 146ng/ul and I am treating RNA every-time after isolation with DNase I. For PCR I am using high fidelity taq i.e., FX-NEO. I am getting amplication in Housekeeping gene (actin) so no-problem in C-DNA quality I guess. Interestingly by mistake I eluted that 3kb band and cloned it in Plant transformation vector successfully.
So I dont know what should I do next. Should I need to design the primer again or just try the PCR of rice genomic DNA using same primer. Please guide me
Thank you
Regards
Harshraj
As suggested the 3 kb amplicon might contain the introns from genomic DNA amplification. Is there any possibility of alternative splicing producing a cDNA from a bigger mRNA of the same gene?
Are the two primers you used at the two ends of the expected gene or are they internal sequences? Try amplifying the genomic DNA with the same primers and see if you get the 3 kb DNA Band.
I am using the two end primers as I want to isolate the full length gene. anyways Thank you for your suggestion I will try PCR of genomic DNA
Maybe genomic DNA contamination. Keep PCR with cDNA and genomic DNA that may resolve your query.
Hi Harshraj,
1. cDNA is made from mRNA. Abundant mRNA makes good cDNA. Some mRNA are tissue-specific and some are 'development-stage'-specific. In other words, they will only be produced from that tissues and at a duration of plant development. Has the profile of mRNA transcript level of this specific gene been studied in the rice variety you are studying, and know that when and where the mRNA will be transcribed (usually by Northern Assay)? If so, did you isolate total RNA from that specific tissue and at the right developmental stage of plants (especially the stage with maximum mRNA transcript of your gene)?
2. For one example, see the attached paper Fig.1., which depicts different carrot sugar-digesting enzyme mRNA levels at different tissues and different plant developmentaf stages (weeks).
3. If you just want to overexpress the gene under different promoter (such as 35S), you can either use cDNA or its genomic DNA (ORF) for that purpose. You said you had successfully cloned that 3-kb DNA fragment into a plasmid. You might as well try it anyway, but make sure the DNA fragment is your gene and its sequence is correct. I had done that myself (using ORF) and it succeeded.
Good luck on your research.
As suggested, the expression of mRNA being tissue and stage specific, you have to ensure that the right mRNA is used to construct cDNA. Any luck with genomic DNA amplification?
Thank you Dr. Yuan for your response. My gene was salinity responsive so it express only after salinity stress. So I gave the salinity stress to rice plants and then i was able to isolate the gene after that t I cloned the 1.4kb C-DNA fragment successfully.
Thank you once again I always reads your answer even if you answer to other researchers also. your answers are always informative and interesting.
I hope you will guide me in future also.
Good luck to your future research.
Thank you !
Dr. Pranab thank you for your response. I got C-DNA isolated after Salinity stress treatment. soi learned that this gene express only under salinity stress condition.
Hi Harshraj,
Thanks for your feedback, and congratulations on getting your right cDNA!!
Good luck on your research too!
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