difficulty synthesising cDNA from total RNA - (Apr/09/2008 )
Hello,
Yes, did a negative control PCR (no template, just water). The result was negative so I'm pretty sure the positive result in the -RT reaction is not due to contamination in my PCR materials. As for the second experiment, it's possible that I made a mistake but not likely because most of my previous attempts at reverse transcription also failed to produce cDNA.
[/quote]
Hmm...very confusing. Perhaps you should start over from the begining. Isolate your RNA, do an RT reaction - but leave out the DNase and RNase treatment ( I honestly don't think you should need an RNase treatment anyway). Do your RT reaction with oligodT and use your B-actin primers. If this reaction works - you get both genomic DNA and the cDNA, and your negative (water) control is clean, then try to do the DNase treatment. I would recommend to column purify your RNA after the DNase treatment - qiagen sells some columns that are nice for this, called minElute. I think these columns are more efficient at removing DNase than doing a precipitation, and in the end you don't have to worry about whether you got your pellet resuspended nicely, etc.
As for why you are getting a positive result in your -RT reaction, it very well could be the case that you still have genomic DNA present. I typically use Qiagen's RNA extraction kits, and I found that if I did an on column DNase treatment, that I still had genomic contamination. However, if I went through the entire purification process, then did the DNase treatment, and did a second column purification the genomic DNA was completely gone. I realize that you are doing a completely different protocol, but if your RNA is not very pure to begin with (as mine was on the column), then its possible that your DNase treatment isn't working very well because there are other things present that are interfering with it. In any case, I would get the RT-PCR working without the DNase treatment first so that you at least know that you're doing that much of the protocol right. Then try to tackle the DNase treatment.
-smu2-
Hi Smu,
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
-calavera1984-
QUOTE (calavera1984 @ Apr 22 2008, 09:00 PM)
Hi Smu,
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
That's great! It sounds like you're making progress... How many cycles did you run? I would try another PCR reaction, increasing the number of cycles (start with an increase of 5 cycles), or you could try using just a little more cDNA (~1.3 µl instead of 1µl... I assume you meant this is undiluted cDNA from your RT reaction), or you could try both. Also, sometimes adding DMSO at 5% of the reaction volume can really help improve amplification. PCR often takes a little tweaking to get just right, but it definitely sounds like you're on the right track!
-MolBioGirl-
QUOTE (MolBioGirl @ Apr 22 2008, 07:02 PM)
QUOTE (calavera1984 @ Apr 22 2008, 09:00 PM)
Hi Smu,
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
That's great! It sounds like you're making progress... I would try another PCR reaction, increasing the number of cycles (start with an increase of 5 cycles), or you could try using just a little more cDNA (~1.3 µl instead of 1µl... I assume you meant this is undiluted cDNA from your RT reaction), or you could try both. PCR often takes a little tweaking to get just right, but it definitely sounds like you're on the right track!
Hello. Good to see you here again MolBioGirl
You've got great suggestions as usual. I didn't purify the cDNA at the end like most of the previous suggestions. Seems to work a bit better.Yupyup....undiluted cDNA.
Ok. I'll try both. I guess that means PCR conditions are not the same when amplifying from cDNA? If so, do I need to do gradient and optimise Mg concentrations again or do those remain the same?
-calavera1984-
QUOTE (calavera1984 @ Apr 22 2008, 10:07 PM)
QUOTE (MolBioGirl @ Apr 22 2008, 07:02 PM)
QUOTE (calavera1984 @ Apr 22 2008, 09:00 PM)
Hi Smu,
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
That's great! It sounds like you're making progress... I would try another PCR reaction, increasing the number of cycles (start with an increase of 5 cycles), or you could try using just a little more cDNA (~1.3 µl instead of 1µl... I assume you meant this is undiluted cDNA from your RT reaction), or you could try both. PCR often takes a little tweaking to get just right, but it definitely sounds like you're on the right track!
Hello. Good to see you here again MolBioGirl
You've got great suggestions as usual.Ok. I'll try both. I guess that means PCR conditions are not the same when amplifying from cDNA? If so, do I need to do gradient and optimise Mg concentrations again or do those remain the same?
Aw thanks -- I'm just glad to help.
Your other PCR conditions should remain the same (temperatures, times, Mg, etc.), but in my experience, it's usually much easier to amplify fragments from very small amounts of genomic DNA (or plasmid) than from cDNA. I usually have to add a little bit more cDNA to start with... Good luck! Let us know how it turns out.
-MolBioGirl-
QUOTE (MolBioGirl @ Apr 22 2008, 07:13 PM)
QUOTE (calavera1984 @ Apr 22 2008, 10:07 PM)
QUOTE (MolBioGirl @ Apr 22 2008, 07:02 PM)
QUOTE (calavera1984 @ Apr 22 2008, 09:00 PM)
Hi Smu,
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
That's great! It sounds like you're making progress... I would try another PCR reaction, increasing the number of cycles (start with an increase of 5 cycles), or you could try using just a little more cDNA (~1.3 µl instead of 1µl... I assume you meant this is undiluted cDNA from your RT reaction), or you could try both. PCR often takes a little tweaking to get just right, but it definitely sounds like you're on the right track!
Hello. Good to see you here again MolBioGirl
You've got great suggestions as usual.Ok. I'll try both. I guess that means PCR conditions are not the same when amplifying from cDNA? If so, do I need to do gradient and optimise Mg concentrations again or do those remain the same?
Aw thanks -- I'm just glad to help.
Your other PCR conditions should remain the same (temperatures, times, Mg, etc.), but in my experience, it's usually much easier to amplify fragments from very small amounts of genomic DNA (or plasmid) than from cDNA. I usually have to add a little bit more cDNA to start with... Good luck! Let us know how it turns out.
I could kiss you right now MolBioGirl!!!!
I used 1.3ul undiluted cDNA and increased the cycles to 40. A perfect band! And it's of the correct size!! However, there's still a slight concern.
As in my first post, I cannot afford to have genomic DNA in my sample because one of my primer sets targets an intron (it's been shown to exist in one of the transcripts I'm looking for. That's why I cannot afford to have genomic DNA give false positives if that particular transcript is absent).
Anyway, my +RT shows the correct cDNA band ONLY. My -RT reaction however shows the genomic band ONLY (but very much less than the cDNA). I'm assuming the DNase step did not go as planned. Any suggestions there?
*KISS YOU*
-calavera1984-
QUOTE (calavera1984 @ Apr 23 2008, 02:57 AM)
QUOTE (MolBioGirl @ Apr 22 2008, 07:13 PM)
QUOTE (calavera1984 @ Apr 22 2008, 10:07 PM)
QUOTE (MolBioGirl @ Apr 22 2008, 07:02 PM)
QUOTE (calavera1984 @ Apr 22 2008, 09:00 PM)
Hi Smu,
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
Good suggestion. I might try it. However, I see some glimmer of hope from my most recent RT attempt.
1. My +RT PCR with B-actin primers are positive and the -RT PCR with B-actin primers is negative.................so far so good
2. I tried PCR with my test primers................very very very faint band but is of the expected size. The signal still remains the same using 1ul cDNA in 20ul reaction and 1ul cDNA in 50ul reaction. The signal is far better when I try it on genomic DNA though.
That particular transcript is abundant in the tissue I investigated. Therefore, any suggestions as to how I can improve on those results? Should I try RT again using more RNA or is it a simple PCR issue or what?
That's great! It sounds like you're making progress... I would try another PCR reaction, increasing the number of cycles (start with an increase of 5 cycles), or you could try using just a little more cDNA (~1.3 µl instead of 1µl... I assume you meant this is undiluted cDNA from your RT reaction), or you could try both. PCR often takes a little tweaking to get just right, but it definitely sounds like you're on the right track!
Hello. Good to see you here again MolBioGirl
You've got great suggestions as usual.Ok. I'll try both. I guess that means PCR conditions are not the same when amplifying from cDNA? If so, do I need to do gradient and optimise Mg concentrations again or do those remain the same?
Aw thanks -- I'm just glad to help.
Your other PCR conditions should remain the same (temperatures, times, Mg, etc.), but in my experience, it's usually much easier to amplify fragments from very small amounts of genomic DNA (or plasmid) than from cDNA. I usually have to add a little bit more cDNA to start with... Good luck! Let us know how it turns out.
I could kiss you right now MolBioGirl!!!!
I used 1.3ul undiluted cDNA and increased the cycles to 40. A perfect band! And it's of the correct size!! However, there's still a slight concern.
As in my first post, I cannot afford to have genomic DNA in my sample because one of my primer sets targets an intron (it's been shown to exist in one of the transcripts I'm looking for. That's why I cannot afford to have genomic DNA give false positives if that particular transcript is absent).
Anyway, my +RT shows the correct cDNA band ONLY. My -RT reaction however shows the genomic band ONLY (but very much less than the cDNA). I'm assuming the DNase step did not go as planned. Any suggestions there?
*KISS YOU*
I'm glad it worked for you.. that's great!Well, DNAse treatments do not always remove ALL DNA from the sample -- there still may be trace amounts left, and since you've had to increase to 40 cycles of PCR, you will amplify the small amount that is there. For the most part, it seems your conditions are worked out. SMU2 is right -- now that you've got the PCR conditions worked out and you know your RT worked fine, you can try again, this time taking your RNA through a more rigorous DNAse treatment.(Follow the instructions for whatever DNAse enzyme you are using). I agree with SMU2 also that you should not need to do an RNAase treatment. Just take one thing at a time. Good luck!
-MolBioGirl-