Hi,

First I recommend you read the link below to get information about cDNA synthesis. Based on my personal experience, I recommend to use Takara or Thermo Scientific kits.

https://www.thermofisher.com/us/en/home/life-science/pcr/reverse-transcription/5steps-cDNA.html

Hi,

I'm not a professional in cDNA synthesis, but I've done it several times.

Here is another guide (almost the same as Marzieh Rostaminejad recommended).

https://www.thermofisher.com/br/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/rt-education.html

It's importante to have a RNA sample with good integrity and purity. So, before cDNA synthesis try to quantify your samples in nanodrop and check the integrity by electrophoresis on a denaturing gel. A good RNA sample wil have a 260/230 ratio ranging 2.0 - 2.2 and 260/280 ratio ranging 1.8 - 2.0. And for good integrity you will have the intensities of the 28s and 18s bands in the gel at a ratio of 2:1 respectively.

I've been using the High-Capacity cDNA Reverse Transcription Kit from ThermoFisher and have never had any problems with it. Probably the kit comes with a protocol containing volumes and concentrations, and if not, you can find it in the website.

A very good discussion here is about the amount of RNA. Surely the manufacturer will also provide this information and it's good to follow this. But you can find people using from 100ng till 5ug or even less or more.

And about troubleshooting, try the links we left here or look for questions and discussions like yours here in researchgate. I find almost all the solutions to my problems here.

Hope this helps!

Hello Ahmed Abdelhamid

You can isolate total RNA (a mix of ribosomal RNA, transfer RNA and mRNA) from tissues and cells.

For tissues, homogenize tissue samples in 1 ml of TRIZOL reagent per 50 to 100 mg of tissue using a homogenizer. TRIZOL reagent maintains the integrity of RNA due to highly effective inhibition of RNase activity while disrupting cells and dissolving cell components during sample homogenization The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for homogenization.

For cell monolayer, rinse the cells with ice cold PBS once. Lyse cells directly in culture dish/ plate by adding TRIZOL Reagent and scraping the cells with cell scrapper. For suspension cells, spin the cells for 5 min at 300 X g. Remove media and resuspend cells in ice cold PBS. Pellet cells by spinning at 300 X g for 5 min. Lyse cells with TRIZOL Reagent. Use 1 ml of the TRIZOL reagent for 5-10 million cells.

Incubate the homogenized samples (tissue or cells) for 5 minutes at room temperature. Centrifuge to remove cell debris. Transfer the supernatant to new tube.

To carry out phase separation, add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Vortex samples vigorously for few secs and incubate at room temperature for 5 minutes. Centrifuge the samples at 10,000 x g for 15 minutes at 4 degree C. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully into fresh tube.

Precipitate RNA from the aqueous phase by mixing with 500ul isopropyl alcohol. Incubate samples for 10 minutes at room temperature, and centrifuge at

10,000 x g for 10 minutes at 4 degree C. The RNA precipitate will form a gel-like pellet on the side and bottom of the tube. Wash the RNA pellet once with 75% ethanol. Vortex and centrifuge at 7,500 x g for 5 minutes at 4 degree C.

Air-dry RNA pellet for 5-10 minutes. Do not let the RNA pellet dry completely as this will greatly decrease its solubility. Dissolve RNA in DEPC-treated water and take the OD reading to determine sample concentration and purity.

A 260/280 ratio of ~2.0 is generally accepted as pure for RNA and generally acceptable 260/230 ratios are in the range of 2.0 – 2.2.

Also, run an agarose mini gel. The 2 ribosomal RNA bands (18S and 28S) should be visible under UV light in the presence of ethidium bromide which is a good indication that the RNA sample in intact (not degraded by RNase).

Finally, you can synthesize cDNA using a commercially available kit. You can use 500ng -1ug RNA for cDNA synthesis. But this will depend on the cDNA synthesis kit that you use. Please follow the instruction provided in the kit insert. The resulting cDNA can be stored for later analysis of gene expression at -20°C in aliquots to avoid repeated freeze/thaw cycles.

Precautions to be taken:

When working with RNA it is necessary to be extremely careful. Wear gloves at all times. Make sure that all solutions, tubes, pipet tips, etc. are for RNA work. The bench or other surface has to be RNase-free.

Good Luck.