difficulty synthesising cDNA from total RNA - (Apr/09/2008 )

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?

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?

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!

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?

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?

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?

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.

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?

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*