New to RNA... Does this cDNA gel look OK? - (May/20/2008 )
Hi All,
I am a graduate student studying molecular systematics. I would like to use nuclear protein-coding genes for animal phylogenetics. I successfully extracted RNA from a few molluscs and today I tried to perform a reverse transcription. I have never seen a gel of "good" cDNA so I have no idea if this looks like it should. I guess I was expecting a big smear instead of what appears to be two somewhat distinct bands. Does the single-stranded DNA fold up to make a mess like this?
I used Promega's ImProm-II RT with polyT primers and 1 ul of extract from from living tissue that was extracted using an RNeasy kit (so it should have been a lot of RNA). For the gel I mixed 3 ul of putative cDNA with 3 ul of green/orange loading dye and put 5 of that into the wells. I adjusted the levels of the image in photoshop so that is why it looks like I used a liter of ladder.
Any comments / advice would be very much appreciated.
Thank you!
Kevin
I am a graduate student studying molecular systematics. I would like to use nuclear protein-coding genes for animal phylogenetics. I successfully extracted RNA from a few molluscs and today I tried to perform a reverse transcription. I have never seen a gel of "good" cDNA so I have no idea if this looks like it should. I guess I was expecting a big smear instead of what appears to be two somewhat distinct bands. Does the single-stranded DNA fold up to make a mess like this?
I used Promega's ImProm-II RT with polyT primers and 1 ul of extract from from living tissue that was extracted using an RNeasy kit (so it should have been a lot of RNA). For the gel I mixed 3 ul of putative cDNA with 3 ul of green/orange loading dye and put 5 of that into the wells. I adjusted the levels of the image in photoshop so that is why it looks like I used a liter of ladder.
Any comments / advice would be very much appreciated.
Thank you!
Kevin
Hi Kevin!
I just successfully completed an RT-PCR assay myself, but not without a few failures! Here's my advice to you:
1) You need to include a negative control for each RNA extraction you do. There is NO way to tell if the bands you saw are from cDNA amplification or simply DNA contamination in your RNA preps. The negative control that you must include is, following the protocol for synthesizing cDNA, but when you get to the step where you add reverse transcriptase, add the enzyme to one sample, and RNase-free water to the other. By doing this, you should expect to see no band for your no-RT control when doing the PCR. If you see a band in your no RT control, then that means you have contaminating DNA in your preps.
2) I highly recommend you use Qiagen's RNeasy mini kit and the Qiashredder spin columns (sold separately) which will homogenize your lysates.
3) Also, purchase RNase-free DNase I (preferably from Qiagen should you use their kit) and perform an on-column DNA digestion.
4)* Should the above suggestions still not work for you, there is one other thing you can do which I have had success with. It's a little out there, but it does work!
After eluting purified RNA, take about 10uL of the eluate and restriction digest it with a highly frequent cutter such as DpnI. Use only newly purchased enzyme and buffer, and only digest for 30 minutes max. Following the digestion, you would have to clean up the RNA and perform another on-column DNA digestion. That can be done using the Qiagen kit. The purpose of this is to gobble up any contaminating DNA in your RNA preps, which would make it easier to get rid of in the clean-up process.
I hope this helps!
hi,
RT-PCR gels look like any other gels you'd expect to see from regular PCRs. Your gel looks fine. Just make sure that the bands you see correspond to the size of the cDNA and not gDNA. I'm assuming you've already tested your PCR primers on gDNA beforehand and obtained a larger molecular weight band. Send your RT-PCR products for sequencing and see if it matches your cDNA sequence to be sure.