I have RNA, isolated from Yeast cells (By TRI (SIGMA) reagent), quality is good after Dnase-1 treatment (on Gel), but cDNA is not formed. MMLV RT is working fine for other strains (RNA samples). PCR with genomic DNA is fine. PCR for all other transcripts (Actin, hsp60, tfiid etc not working), Any reason? Any inhibitor of RT, if known , please suggest.
Try performing an inhibition curve. Take a 10fold dilution (8 points) of your RNA and do RT of all your dilutions. If you don't obtain a straight line than you might have an inhibitor present.
Based on the provided information it sounds like either residual phenol or alcohol. What were the 260/280 ratios after DNA digestion? What DNAse are you using and how are you removing it from your RNA samples? DNAse contamination will also cause similar problems
phenol present in tri-reagent may cause inhibition to RT enzyme, purify RNA sample prior to cDNA preparation.
Try performing an inhibition curve. Take a 10fold dilution (8 points) of your RNA and do RT of all your dilutions. If you don't obtain a straight line than you might have an inhibitor present.
Most likely culprit is your DNase treatment. Ensure that the DNase is fully inactive, or simply try a TRI reagent extraction separating the RNA from DNA without DNase treatment. Other than that, phenol contamination is a issue typically if incorrect ratios are used in extractions or if small amounts of organic/interphases are inadvertently isolated with aqueous. Guanidine can inhibit RT as can excess protein (typically protein is not an issue when using TRI reagents though).
How do you say that your cDNA is not present? is it by PCR? I would recommend using different amounts of the reverse transcribed product and also have a positive control always in your PCR. This might work if you do not have any inhibitors of PCR in your RNA or the cDNA.
Thankyou all for ur valuable suggestions. I isolated fresh RNA, started afresh and things are fine now.
hey i extracted rna from whole larvae and tried to perform cdna as i need to make rna probes for insitu hybridization, but when i did RT using iscript cdna synthesis kit amd ran the sample on the 1 percent agarose gel, i dont have a band. also before cdna synthesis, i ran the total rna on a gel and i could see the 28 and 18 s bands clearly. could guys tell me what and where have i gone wrong or why cannot i see cdna band on the gel after RT?
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