PCR & Western Blot sample preperation - (Jan/14/2015 )

Hello,

I am VERY new at leaning PCR and western blotting. I have jumped in at many different times during both processes and am confused as to what needs to be done when and for which. My questions are:

1) In general, how would you prepare a sample of cancer cells to perform Reverse Transcript PCR and then qPCR?

2)In general, how would you prepare a sample of cancer cells to run a western blot?

I am especially confused as to when I have sonicate the samples, when to use loading buffer, DTT, and when RNA isolation is needed.

 I know these questions may require long answers, but it would help me immensely.

Thanks!

Cancer cells -> RNA -> cDNA -> RT-PCR and qPCR

Cancer cells -> cell lysate -> western blot

Don't be afraid to ask your lab colleagues. This kind of very basic questions are best addressed that way. Reading helps too.

CPRES on Wed Jan 14 23:48:50 2015 said:

Cancer cells -> RNA -> cDNA -> RT-PCR and qPCR

 

Cancer cells -> cell lysate -> western blot

 

Don't be afraid to ask your lab colleagues. This kind of very basic questions are best addressed that way. Reading helps too.

Thanks again! I do need to be more willing to ask my lab colleagues (I struggle with social interactions at times and have a hard time hearing and interpreting some people who are new to the english language). It also helps me to see it written out on here and it gives me more time to process the information (usually I think of questions during an experiment and ask them and then forget bits if I can not write it down). I am fresh out of Uni into an entry level position and really appreciate any advice.

I've made a long post which I accidentaly deleted, so only in brief.

What type of cancer cells?

The most important thing for qPCR analysis from tissues is preserving the source material from the very start, or your RNA degrades. Either snap froze in liquid nitrogen, or using RNA-later or other fixatives. And subsequently taking care about RNA stability in every further step, preferable using it as soon as possible.

Since it is really a complex topic, you may start with the whole design, what you wan to detect what genes to select, what reference genes and controls to use. You can do that with a piece of paper and computer.

Then take care of the tissue handling, because most probably you won't have all the material at once and would need to store it for some time, so you need to solve this. Also you need to know how you are going to isolate RNA from it at this point, because storage had to be compatible with the isolation procedure.

Other steps (RNA isolation, DNase treatment, RT, qPCR) can be tested and done more on-the-fly.

I actually work with various types of cells (and tissue) depending on the experiment (which is partially why I was getting confused). It is all coming together now though. We have everything at my facility, including -20, -80, and liquid nitrogen. The cells are typically treated with RLT-BME and then stored in the -80 until I have the time to run the RNA isolation, RT, & qPCR. I isolate the RNA using an RNeasy kit.

Well there are some differences disrupting and/or isolation RNA from different tissues, but if you read the complete RNeasy manual it's a good start. They mention many specific problems you may encouter and suggest how to solve them. Supposely you also have a homogenizer, that goes well with the column kit to crush the frozen tissue without degrading the RNA.