CORRECT PCR Incorrect RTPCR - (Nov/10/2009 )
LAK on Nov 11 2009, 03:01 PM said:
DRN on Nov 11 2009, 12:04 PM said:
LAK on Nov 11 2009, 07:05 AM said:
almost a doctor on Nov 10 2009, 05:34 PM said:
Hey, think of the differences between PCR and RT-PCR and you'll get step closer to find out what's wrong:
1. How do you prepare your RNA? Is it good enough? Is there enough?
2. How do you prepare your cDNA? Is the RT reaction efficient enough?
3. How are your primers design? Should they really work on cDNA?
4. Are you using enough/too much cDNA on your PCR?
....
1. How do you prepare your RNA? Is it good enough? Is there enough?
2. How do you prepare your cDNA? Is the RT reaction efficient enough?
3. How are your primers design? Should they really work on cDNA?
4. Are you using enough/too much cDNA on your PCR?
....
You are correct friend.
1. My RNA is good. I hope it is enough. cause i get band in a 50µl reaction. But for 25µl reaction, there is no band. also i tried vortexing after adding RNA and primers into mix (before adding enzymes). still no result.
2. well my cDNA. i use qiagen's one step RT. I do not have access to buy two step or any other reagent. Could you please suggest me if there is a way to find out if my RT reaction is efficient enough. Because even I doubt this.
3. Primers are good. They do work on cDNA.
4. Again, how do I find out. What will happen if there is too much cDNA. Please explain.
LAK,
hv u tried out the RT reaction with control set of primers????
DRN,
how do i do a RT. please explain. after RT what do i do with cDNA.
please consider me a beginner and explain in detail...... (am not an expert as you all are
)i use the invitrogen kit for one-step RT n i take 1ug of RNA. after RT, i add RNAseH. this is required only if u hv to get the RT-PCR product sequenced/cloned (coz u don't want traces of RNA in that case). Next step is to determine
a) cDNA is free of genomic DNA. for that can run a PCR with -RT reaction. by that I mean, the reaction should hv ur RNA but not the cDNA. if u get amplification in this case, it means that there is genomic DNA contamination.
another point to see is if cDNA is fine. for that I do a PCR with primers for costitutive/good-expressing genes......the reaction almost without fail should work for these, if ur cDNA is good.c) next is, ur RNA yield might be good. but what if, the no. of trancripts of ur desired gene is less? what u quantitate is the entire mRNA pool, not ur specific one, right?.......therefore, if u aren't getting the RT-PCR to work, it could also be because u might just hv to use more of the cDNA, more primers........I mean u will hv to standardize that.
hope this is of at least some help........lemme know.
all the best
-DRN-