Hi Noha

working with RNA is bit tricky since it requires a lot of precautions from sample preservation to RT PCR. You need a clean dust free environment for extraction, certified DNase/RNase free plasticwares and glasswares and good laboratory practices to avoid contaminating your RNA samples with RNases. 

In our lab, we extract RNA from a number of samples including serum, CSF, tissue, plants, small whole animal etc. using TRI reagent, followed by re-purification (if required) using Qiagen RNAeasy mini kits. They work fine with us. We then usually run the samples on denaturing agarose gels to assess the quality of the extracts and then quantify on a Picodrp UV spectro and go for RT-PCR. 

I am attaching some guidelines to work with RNA. Hope you find them useful..

Best Wishes,

Sib

Hi Noha

working with RNA is bit tricky since it requires a lot of precautions from sample preservation to RT PCR. You need a clean dust free environment for extraction, certified DNase/RNase free plasticwares and glasswares and good laboratory practices to avoid contaminating your RNA samples with RNases. 

In our lab, we extract RNA from a number of samples including serum, CSF, tissue, plants, small whole animal etc. using TRI reagent, followed by re-purification (if required) using Qiagen RNAeasy mini kits. They work fine with us. We then usually run the samples on denaturing agarose gels to assess the quality of the extracts and then quantify on a Picodrp UV spectro and go for RT-PCR. 

I am attaching some guidelines to work with RNA. Hope you find them useful..

Best Wishes,

Sib

To work with RNA, you have to keep in mind that first of all, you have to destine (if it's possible) certified DNA/RNA free glassware (even autoclaved) and a workstation to prepare the mixes for the reverse transcription (if you're using a two step qPCR), in the workstation a UV-light works really well to keep the environment RNA free previous the RT. This workstation has to be used only for the reverse transcription and once you have cDNA, you can prepare the mastermix for the PCR in a normal laminar flow cabinet. So, a separate workstation for the RT and other workstation for the PCR is pretty recommended.

When you're extracting the RNA, you have to work under a hood because of the chemicals used during the extraction such as beta-mercaptoethanol which is really toxic. Additionally, all your samples have to be in chilled ice in order to avoid any action of RNAses present in the sample itself, also the whole process has to be done on tubes in chilled ice. Qiagen kits tend to work pretty well and you can use a specific kit depending on the origin of your sample (animal, human or plan tissue or even bacteria). Furthermore, at the time of the elution, if you know or pressume that your sample has low concentration of your target RNA, you can use less volume to elute the RNA in the column, so if the protocol tells you to use 50 uL, it's better to use 30 uL and also let stand the certified RNAse free water (it's better to use this water rather than any buffer in the kit for downstream applications) in the column filter during 2-4 mins before centrifuging twice (add 30 uL to the column, 4 mins, centrifuge, take the same water and put in the same column during another 4 mins and finally centrifuge).

During the RT, you can use RNAse inhibitors such as RNAsin to get better results, also always try to keep the RNA samples in -70°C for months (sometimes up to 6 months) and the cDNA in -20°C for days (it depends on your sample, but it could be used up to 2 weeks). In addition, do aliquots of all your work solutions (specially DNA, RNA, primers, enzymes, so on) in order to avoid thawing because every time that you thaw your samples you're losing DNA (specially RNA) and the work capacity of your solutions which need refrigeration. Always make sure to eliminate any ethanol residue through additional centrifugations with a new collection tube and standing for a couple of minutes in 40°C (when you have DNA).

Hope that this information is useful for you.

Cheers.

THANKS FOR ALL. I WANT KNOW What is the better (triazole or column spin ) for rna rxtarction?

In our company we use kits from jane bioscience for bacterial RNA extraction. We are satisfied with their simple protocol and consistent results. You may try this http://www.jenabioscience.com/cms/en/1/catalog/1601_rna_purification.html

if you want more quantity go for Trizol method but if you want quality go for Column method. if your handling is good you could get nice quality in Trizol method also.

for the precautions:

1. perform in low temperature.

2. treat your glass/lab/plastic wares,  with DEPC

3. use molecular grade water.

4. always wear gloves.

5. don't be greedy while removing aqueous phase as this may lead to poor quality.

All the best

It depends on you budget, you can use:

1- Trizol method (Cheaper)

2- Kits such as (RNA extraction kit) from Qiagene or invetrogen (easier and faster)

Both methods are good. However, Trizol give you more RNA yeild

Depending on what you're working with, it's better one option (taqman or syber green) over the other, you can watch this seminar 

https://www.youtube.com/watch?v=qLAGD-Wy0D8

it's pretty good explaining this.

Hi Noha,

All the suggestion above are OK. But before you even want to purchase any of them, you have to consider the sample size first. Then choosing the best suitable kit.

You can also check out a review article on this topic by Labome. https://www.labome.com/method/RNA-Extraction.html

It depends entirely on your purpose and your sample size.

you can also check out this article

Sibnarayan Datta : After the RNA isolation using Triazole, do you directly load RNA into column or process it further?? Please share the protocol as to how do you further purify RNA. I have isolated RNA with A260/A280 1.9 value, I want to further purify it with Qiagen Mini column