difficulty synthesising cDNA from total RNA - (Apr/09/2008 )
An alcohol precipitation is usually done after DNase treatment to get rid of salts, enzyme, and free nucleotides, all of which can mess with your RT reaction.
A housekeeping gene is very important as a control, and will tell you 1) that your PCR works and 2) that your RNA is good.
Just make sure that the fragment you amplify of your hk gene is different enough in size from your target gene so you can see them both in your ++ reactions. Also make sure to run other reactions with your hk and target genes seperately though, in case the primers interact or if competition favors one product over the other...
You can use up to 5µg RNA in your RT reactions -- maybe you need to increase yours a little.
Also, you really shouldn't need to column purify your PCR product before running on a gel. You shouldn't see anything other than your DNA anyway, and adding extra steps just adds more chances to lose your product...
Hi again,
Regarding primers:
My primers are located in individual exons i.e they don't cross over adjacent exons. The reason being my aim is to determine whether the the particular transcript is expressed or not. If the primers spanned adjacent exons, they would not bind to genomic DNA during PCR even if I tested them on it. Therefore, if the RT worked but the transcript is not expressed......no product. If the RT worked, transcript is expressed, but the primers don't anneal........no product also. I hope that explains things better. Correct me if I'm wrong.......very confused already.
OK. Alcohol precipitation sounds good. So let's say I've done my DNase treatment, I now have a tube with the sample in 10ul of whatever buffers and reagents, enzymes, etc. How do I go about doing it step by step. Sorry, I'm very new to this.
OK. Will skip the purification step.
Thanks a lot. Really appreciate it.
Hi again,
Regarding primers:
My primers are located in individual exons i.e they don't cross over adjacent exons. The reason being my aim is to determine whether the the particular transcript is expressed or not. If the primers spanned adjacent exons, they would not bind to genomic DNA during PCR even if I tested them on it. Therefore, if the RT worked but the transcript is not expressed......no product. If the RT worked, transcript is expressed, but the primers don't anneal........no product also. I hope that explains things better. Correct me if I'm wrong.......very confused already.
[/quote]
To clarify: If your sequence is: exon1 - intron1 - exon2, and your primers are in exon1 and exon2 then your cDNA will be a different size than your genomic DNA, enabling you to determine if there is genomic DNA present. If you can design primers like this, then you don't necessarily need to do a DNase step because you can tell your products apart. If your primers are both in the same exon - then your cDNA and genomic DNA will give you the same size band so you won't be able to tell them apart - you will need to do a DNase step. From your description, if your have a primer that is part of exon1 and part of exon2, then you are correct in your interpretation - provided of course that annealing goes as planned. Some funny things can happen if you design primers like that for RTPCR. Hope this helps.
Regarding primers:
My primers are located in individual exons i.e they don't cross over adjacent exons. The reason being my aim is to determine whether the the particular transcript is expressed or not. If the primers spanned adjacent exons, they would not bind to genomic DNA during PCR even if I tested them on it. Therefore, if the RT worked but the transcript is not expressed......no product. If the RT worked, transcript is expressed, but the primers don't anneal........no product also. I hope that explains things better. Correct me if I'm wrong.......very confused already.
OK. Alcohol precipitation sounds good. So let's say I've done my DNase treatment, I now have a tube with the sample in 10ul of whatever buffers and reagents, enzymes, etc. How do I go about doing it step by step. Sorry, I'm very new to this.
OK. Will skip the purification step.
Thanks a lot. Really appreciate it.
No problem -- don't feel bad about asking questions. That's what this forum is for!
Okay -- SMU is right. What I actually meant about the primers spanning an intron was that if you have "exon1-intron-exon2", you could have your forward primer in "exon1" and your reverse primer in "exon2"...(or 3, 4, etc... to get your desired fragment size). That way, if the gene is amplified from genomic DNA, you will see the entire fragment that includes the intron. If it's amplified from cDNA, the fragment length will only include the exons. This way, you should be able to tell them apart.
I agree with SMU on this also -- If you design primers that include parts of different exons within one primer, sometimes you don't get the desired result. (Likely because many mRNA's have more than once splice form).
On alcohol pptn: There are several different methods to use, and many people have their own preferences regarding what works the best. But one of the most popular (and tried and true, as far as I'm concerned) is the standard ethanol precipitation. Even for this, there are variations, depending on preference. Some people use sodium acetate or LiCl... I use the ammonium acetate method:
-add 1/10th volume 5M ammonium acetate
-add 2.5 volumes cold 95% ethanol
-precipitate at -20° or -80° for at least 30 min. (can leave overnight in freezer)
-spin at 13000 RPM, 15min to pellet RNA
-aspirate supernatant, add ~500µl cold 70% etoh
-spin 13000 RPM ~5 min
-aspirate supernatant, spin again 13000 RPM, 1min
-aspirate supernatant, invert open tube on Kimwipe and allow pellet to air dry ~20 min
-resuspend pellet in nuclease free water or 1xTE (depending on size of pellet, anywhere from 20µl-70µl)
So in your case, starting with your DNAsed solution of 10µl, I would start by adding 80µl of nuclease free water to bring your volume up to 90µl. Then I would add 10µl of 5M ammonium acetate, vortex briefly, then add 250µl of cold 95% etoh. Then put in the freezer to precipitate, and follow the rest of the above steps... Then your RNA will be ready to spec and use.
I hope this helps!
Thanks for that. I'm less confused now
No problem -- don't feel bad about asking questions. That's what this forum is for!
Okay -- SMU is right. What I actually meant about the primers spanning an intron was that if you have "exon1-intron-exon2", you could have your forward primer in "exon1" and your reverse primer in "exon2"...(or 3, 4, etc... to get your desired fragment size). That way, if the gene is amplified from genomic DNA, you will see the entire fragment that includes the intron. If it's amplified from cDNA, the fragment length will only include the exons. This way, you should be able to tell them apart.
I agree with SMU on this also -- If you design primers that include parts of different exons within one primer, sometimes you don't get the desired result. (Likely because many mRNA's have more than once splice form).
On alcohol pptn: There are several different methods to use, and many people have their own preferences regarding what works the best. But one of the most popular (and tried and true, as far as I'm concerned) is the standard ethanol precipitation. Even for this, there are variations, depending on preference. Some people use sodium acetate or LiCl... I use the ammonium acetate method:
-add 1/10th volume 5M ammonium acetate
-add 2.5 volumes cold 95% ethanol
-precipitate at -20° or -80° for at least 30 min. (can leave overnight in freezer)
-spin at 13000 RPM, 15min to pellet RNA
-aspirate supernatant, add ~500µl cold 70% etoh
-spin 13000 RPM ~5 min
-aspirate supernatant, spin again 13000 RPM, 1min
-aspirate supernatant, invert open tube on Kimwipe and allow pellet to air dry ~20 min
-resuspend pellet in nuclease free water or 1xTE (depending on size of pellet, anywhere from 20µl-70µl)
So in your case, starting with your DNAsed solution of 10µl, I would start by adding 80µl of nuclease free water to bring your volume up to 90µl. Then I would add 10µl of 5M ammonium acetate, vortex briefly, then add 250µl of cold 95% etoh. Then put in the freezer to precipitate, and follow the rest of the above steps... Then your RNA will be ready to spec and use.
I hope this helps!
[/quote]
Very helpful as usual. Thanks. Will give it try.
I'm just waiting for my reagents and housekeeping gene primers to arrive before i can proceed further into the RT reaction and PCR.
Fingers crossed very tightly.
Hello again,
I've tried reverse transcription again with DNase and RNase treatments as usual. After that, I did a PCR using the reverse transcription product using Beta Actin primers. In the +RT (reverse transcriptase) reaction, the PCR product gave a band of the expected size (corresponding to cDNA). However the -RT reaction also gave a band of the same size. Any idea why that may be the case?
My assumption is that RNA is still present even after RNase was used.
I've tried reverse transcription again with DNase and RNase treatments as usual. After that, I did a PCR using the reverse transcription product using Beta Actin primers. In the +RT (reverse transcriptase) reaction, the PCR product gave a band of the expected size (corresponding to cDNA). However the -RT reaction also gave a band of the same size. Any idea why that may be the case?
My assumption is that RNA is still present even after RNase was used.
Oh...forgot to mention this....
In a previous batch of cDNA (without DNase and RNase) I got 2 bands from the PCR with Beta Actin primers. One band corresponded to the genomic DNA sequence which I had expected. However the 1 band which corresponded to the cDNA mentioned in my previous post was also present. BUT the cDNA that was used had been made using gene specific primers so I did not expect the lower molecular weight band and yet it was still there.
In regards to the previous post, the cDNA had been made with oligodt primer and DNase and RNase treatment. The higher molecular weight band (corresponding to the genomic sequence) was eliminated but the lower molecular weight band (corresponding to the cDNA.....I hope) remains in both +RT and -RT reactions. Am I still amplifying genomic DNA??
Uh oh......Tried making another batch of cDNA. my beta actin PCR didn't work at all. Very lost now.
I've tried reverse transcription again with DNase and RNase treatments as usual. After that, I did a PCR using the reverse transcription product using Beta Actin primers. In the +RT (reverse transcriptase) reaction, the PCR product gave a band of the expected size (corresponding to cDNA). However the -RT reaction also gave a band of the same size. Any idea why that may be the case?
My assumption is that RNA is still present even after RNase was used.
Oh...forgot to mention this....
In a previous batch of cDNA (without DNase and RNase) I got 2 bands from the PCR with Beta Actin primers. One band corresponded to the genomic DNA sequence which I had expected. However the 1 band which corresponded to the cDNA mentioned in my previous post was also present. BUT the cDNA that was used had been made using gene specific primers so I did not expect the lower molecular weight band and yet it was still there.
In regards to the previous post, the cDNA had been made with oligodt primer and DNase and RNase treatment. The higher molecular weight band (corresponding to the genomic sequence) was eliminated but the lower molecular weight band (corresponding to the cDNA.....I hope) remains in both +RT and -RT reactions. Am I still amplifying genomic DNA??
If you are seeing a product in -RT reaction that is the same size as your predicted cDNA (and genomic DNA is expected to be larger), then it's possible that you have contamination. This is a fairly common problem with PCR. Did you perform a reaction without any template at all? You should with every PCR experiment you do in order to determine if you have contamination occurring. If you see a band there without any template then you know that you have contamination. Contamination arises from having a small amount of DNA present either in your pipettors, your tubes, or in one of your solutions. The best thing to do is clean up everything as much as you can, make fresh stocks of your primers, water, etc., and use barrier tips. I also tell people to use two sets of pipettors - one to set up the PCR reaction, and one for loading your gel.
Given that you have contamination, you might still be able to tell if your RT reaction was working if you notice a significant difference in the amount of signal between your + and - reactions. If there is a lot more signal in the +RT reaction, then its likely that you did produce cDNA. As for your second experiment not producing any product at all, I suspect that it was a simple mistake - you forgot to add something to the reaction.
I've tried reverse transcription again with DNase and RNase treatments as usual. After that, I did a PCR using the reverse transcription product using Beta Actin primers. In the +RT (reverse transcriptase) reaction, the PCR product gave a band of the expected size (corresponding to cDNA). However the -RT reaction also gave a band of the same size. Any idea why that may be the case?
My assumption is that RNA is still present even after RNase was used.
Oh...forgot to mention this....
In a previous batch of cDNA (without DNase and RNase) I got 2 bands from the PCR with Beta Actin primers. One band corresponded to the genomic DNA sequence which I had expected. However the 1 band which corresponded to the cDNA mentioned in my previous post was also present. BUT the cDNA that was used had been made using gene specific primers so I did not expect the lower molecular weight band and yet it was still there.
In regards to the previous post, the cDNA had been made with oligodt primer and DNase and RNase treatment. The higher molecular weight band (corresponding to the genomic sequence) was eliminated but the lower molecular weight band (corresponding to the cDNA.....I hope) remains in both +RT and -RT reactions. Am I still amplifying genomic DNA??
If you are seeing a product in -RT reaction that is the same size as your predicted cDNA (and genomic DNA is expected to be larger), then it's possible that you have contamination. This is a fairly common problem with PCR. Did you perform a reaction without any template at all? You should with every PCR experiment you do in order to determine if you have contamination occurring. If you see a band there without any template then you know that you have contamination. Contamination arises from having a small amount of DNA present either in your pipettors, your tubes, or in one of your solutions. The best thing to do is clean up everything as much as you can, make fresh stocks of your primers, water, etc., and use barrier tips. I also tell people to use two sets of pipettors - one to set up the PCR reaction, and one for loading your gel.
Given that you have contamination, you might still be able to tell if your RT reaction was working if you notice a significant difference in the amount of signal between your + and - reactions. If there is a lot more signal in the +RT reaction, then its likely that you did produce cDNA. As for your second experiment not producing any product at all, I suspect that it was a simple mistake - you forgot to add something to the reaction.
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.