Reverse-transcriptase PCR troubleshoot HELP - (Jun/23/2005 )
QUOTE
I hope that was a misprint. Getting more cDNA than intial amount of RNA is normally unlikely since the cDNA being produced forms a RNA/DNA hybrid with the RNA. Remember this a transcription not an amplification. So its highly unlikely you got 1 ug/ul of cDNA from 1ng/ul of RNA.
If that was a misprint, did you treat you cDNA to get rid of the RNA? How much of the cDNA are you using for your PCR reaction? Try using different amount of cDNA. If your reaction is 20ul try using 5 ul of cDNA, 5ul of 1:2 dilution, 5ul of 1:5 dilution and 5ul of 1:10 dilution. Too much cDNA can cause inhibition of your PCR. The SuperScript II kit usually recommends 50ng to 5 mg of RNA. I typically use anywhere between 500ng-1ug of intial RNA. I then use 5ul of cDNA with no problems. But sometimes I do a dilution with difficult mRNA targets and it helps in 25% of the cases.
It also important to use everything RNase free and/or depc treated. I remember doing a RT-QPCR a while back and i couldn't get anything. I traced it back to some non-nuclease free 15ml conical tubes i used during RNA extraction.
I not sure if there is a way to isolate mRNA from a total RNA extraction.
If that was a misprint, did you treat you cDNA to get rid of the RNA? How much of the cDNA are you using for your PCR reaction? Try using different amount of cDNA. If your reaction is 20ul try using 5 ul of cDNA, 5ul of 1:2 dilution, 5ul of 1:5 dilution and 5ul of 1:10 dilution. Too much cDNA can cause inhibition of your PCR. The SuperScript II kit usually recommends 50ng to 5 mg of RNA. I typically use anywhere between 500ng-1ug of intial RNA. I then use 5ul of cDNA with no problems. But sometimes I do a dilution with difficult mRNA targets and it helps in 25% of the cases.
It also important to use everything RNase free and/or depc treated. I remember doing a RT-QPCR a while back and i couldn't get anything. I traced it back to some non-nuclease free 15ml conical tubes i used during RNA extraction.
I not sure if there is a way to isolate mRNA from a total RNA extraction.
eh heh heh. No it's not a misprint.
As far as I know, we've been pretty careful and I've been spraying RNAseZap everywhere. The one who harvested the RNA may not have been as careful... Our conical tubes are sterile and I use barrier pipette tips, I also use pcr tubes from Molecular BioProducts. It says on the bag RNase and DNase free but non-sterile so I autoclave them before using....
So I guess either the harvesting part or the actual RT-PCR got contamination somewhere?
Is it advisable to just buy a new set of Superscript III kit at this pt? If only the harvested RNA was an issue, then shouldn't the positive HeLa control show up strongly when I rul on a gel as oppose to very weak as I've been getting?
thanks very very much!!!
-k81878-
QUOTE
thanks very very much!!
Well, we are doing the best we can.
-pBluescript-
*I* would not autoclave tubes I wanted to use for RNA work. Think about what goes into your autoclave. The tubes you get from the manufacturer will be much cleaner before they went in than after they come out. Autoclaving does not remove RNAses. Do you really care if they are sterile? Almost certainly not.
-phage434-
QUOTE (k81878 @ Jul 1 2005, 04:47 PM)
QUOTE
I hope that was a misprint. Getting more cDNA than intial amount of RNA is normally unlikely since the cDNA being produced forms a RNA/DNA hybrid with the RNA. Remember this a transcription not an amplification. So its highly unlikely you got 1 ug/ul of cDNA from 1ng/ul of RNA.
If that was a misprint, did you treat you cDNA to get rid of the RNA? How much of the cDNA are you using for your PCR reaction? Try using different amount of cDNA. If your reaction is 20ul try using 5 ul of cDNA, 5ul of 1:2 dilution, 5ul of 1:5 dilution and 5ul of 1:10 dilution. Too much cDNA can cause inhibition of your PCR. The SuperScript II kit usually recommends 50ng to 5 mg of RNA. I typically use anywhere between 500ng-1ug of intial RNA. I then use 5ul of cDNA with no problems. But sometimes I do a dilution with difficult mRNA targets and it helps in 25% of the cases.
It also important to use everything RNase free and/or depc treated. I remember doing a RT-QPCR a while back and i couldn't get anything. I traced it back to some non-nuclease free 15ml conical tubes i used during RNA extraction.
I not sure if there is a way to isolate mRNA from a total RNA extraction.
eh heh heh. No it's not a misprint.
As far as I know, we've been pretty careful and I've been spraying RNAseZap everywhere. The one who harvested the RNA may not have been as careful... Our conical tubes are sterile and I use barrier pipette tips, I also use pcr tubes from Molecular BioProducts. It says on the bag RNase and DNase free but non-sterile so I autoclave them before using....
So I guess either the harvesting part or the actual RT-PCR got contamination somewhere?
Is it advisable to just buy a new set of Superscript III kit at this pt? If only the harvested RNA was an issue, then shouldn't the positive HeLa control show up strongly when I rul on a gel as oppose to very weak as I've been getting?
thanks very very much!!!
Well if it is not a misprint you are working with very little RNA. Since you looking at mRNA which makes up only 1-3% of total RNA. 1ng/ul of total RNA works out to 10-30 pg/ul of mRNA. Knowing that your target mRNA is but a fraction of the total mRNA and you looking at very little target material for PCR. I would suggest re extracting your RNA to get a better concentration or concentrating the RNA you already have (seeing that you mention running 200-400ul on an agarose gel).
"Is it advisable to just buy a new set of Superscript III kit at this pt? If only the harvested RNA was an issue, then shouldn't the positive HeLa control show up strongly when I rul on a gel as oppose to very weak as I've been getting?"
That depends on how much control RNA are you using for your RT reaction. If you are working with the range that the kit recommends you probably do. If you are diluting the control RNA to level of your other RNA maybe not.
-dobbiewalton-