I plan to use actin as a reference gene for RT-PCR in roots and leaves.
I have designed primers for actin in Passiflora edulis using SRA database, did RNA extraction for leaves and roots and using a kit created its respective cDNA. However I failed to see any bands in the gel for cDNA from leaves. Due to this I am unsure of the band I saw in cDNA from roots. I'm uncertain is the band in the roots is actin.
I plan to create cDNA once again for leaves at the moment and run PCR with the same primer set.
some problems which I think caused this is;
1. the primer might not be efficiently binding due to the presence of other actin homologs
2. the cDNA is not efficiently made (hence re-doing this step)
Please if anyone could advise me on what can be some issues here and how I can troubleshoot it
Thank you
I agree with a re-try of the leaf cDNA and actin amplification. If you want to check the identity of the band from roots you could get it sequenced.
As a note, leaves can have lots of secondary compounds that make a good RNA extraction tricky. I had good success with a CTAB column free method
Having been taught RT-qPCR recently myself. I was taught to check imyprimers on gDNA, purify and sequence that to check your primers are on target. Once you confirm that, move to cDNA and your dilutions to get your standard curve. If you're not getting a clean band on the gDNA, you could redesign your primers to get higher specificity. If you're failing on cDNA, maybe try a different housekeeping gene. Depending where this failing I can provide more detail, so feel free to reach out
We use Qiagen RNeasy kit for extraction, Qubit RNA BR and IQ for quality control, NEB Lunascript RT for cDNA synthesis, GoTaq for our gDNA test and Sso Advanced for qPCR. Hope that helps
Hello!
As suggetsed, it is a good idea to sequence the amplicon to confirm the identity of the band and the specificity of the primers. It calls my attention that you see amplification in roots and not in leaves, because usually extraction of RNA from roots is more complicated and less yield is obtained (at least from my experience in wheat). Is the quality and yield from leave and root RNA similar? It can also happen that the reference gene is less expressed in the leaves, you can try to amplify in a less diluted cDNA. It might be even possible that this gene is not expressed in leaves at all.
Thank you all for the advise and discussion so far. I will work with the following information in my research
We use ef1alpha for qRTPCR in passiflora, and ir word quite well. About the RNA you might have contamination by phenolic compounds. Did you check OD and OD ratios? I recommend the invitek kits for plants
Hello Felipe Sarmiento san
I checked for OD ratios and its 1.8 which is acceptable to be free of proteins. As for the use of ef1 alpha as reference gene, could you kindly direct me to the reference you used to find the primer set for this gene. I am currently working on actin gene from Arabidopsis which then with the use of phytozome and NCBI am constructing the nucleotide sequence for Passiflora
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