RNA is the short form of ribonucleic acid. Mostly RNAs
are single stranded. sometimes they form double stranded
loop. RNA is composed of four nucleotide bases (A-adenine,
G-guanine, U-uracil and C- cytosine) on a ribose sugar and
phosphate diaester backbone.
There are 3 main difference between DNA and RNA.
1- most of the time RNA is single stranded.
2- RNA contains uracil (U), where as DNA contains thymine (T).
3- RNA backbone composed of ribose sugar and DNA is composed of deoxyrobose sugar.
30s rRNA
tRNA There are three main types of RNA.
a- Ribosomal RNA (rRNA)
b- Transfer RNA (tRNA)
c- Messenger RNA (mRNA)
There are other types of RNA such as RNAseP RNA, m1RNA, snRNA, uRNA, siRNA, antisense RNA etc. Among all rRNA is the most abundant RNA of every living cell.
RNA extraction:DNA is more stable than RNA. At high temperature RNA cannot stay long and ribonuclease enzyme is ubiquitously present in the cell. So, it is difficult to extract RNA from cell. There are seversl ways to extract RNA from samples, the most common of these is Guanidinium thiocyanate-phenol-chloroform extraction. This method often uses a proprietary formulation of this reagent called Trizol. RNA should be stored at at -80°C.
Wear gloves when handling trizol, work RNase-free!
1. Aspirate medium from cells grown in dishes
2. Wash with 1XPBS
3. In the fume hood, add 4ml of Trizol to cells on plastic of 10cm plate 2.5 ml on 60 mm plate,
1 ml on 35mm plate, add 3 ml on 3 D culture ( 35 mm plate)
With a 10ml pipette, pipette up and down for 2-3 min until solution is no longer viscous
4. Transfer onto a 15 ml polypropylene tube
5. Spin down the EHS samples in Sorvall Centrifuge for 10 min at 9000 rpm at 4 °C ( No dissolved protein will be pelleted), and transfer supernatant into fresh tube
6. Add 20% of Chloroform for Trizol ® ( i.e. 0.2 ml Chloroform for each 1 ml Trizol ® )
7. Close tubes firmly and shake and vortex vigorously for at least 15 seconds
8. Let sit at room temperature for 10 minutes.
9. Centrifuge for 15 min at 9000rpm at 4 °C
10. Transfer upper, aqueous phase to fresh tube, avoid transfer of ANY interface!!
11. Add 0.5 volume IPA to the clean supernatant (i.e. 0.5 ml IPA for each ml Trizol) and add 0.5 µl Glycogen for each ml Trizol
12. Mix immediately by inverting tubes 5-8 times
13. Incubate at room, temperature for 5-10 min.
14. Centrifuge at 9000 rpm for 10 min, 4 °C
15. Discard supernatant
16. Washing with 1ml of 75% EtOH (RNase –free) to each tube
17. Resuspend dry pellet in nuclease-free water and keep on ice
EHS samples in 20-30 µl water each
Plastic samples in 60-80
See also,
http://en.wikipedia.org/wiki/RNA
http://www.lbl.gov/lifesciences/BissellLab/labprotocols/rnaextraction.htm
http://www.lebs.cnrs-gif.fr/golinelli/golinelli_eng.htm
http://www.aps.anl.gov/Science/Highlights/2001/ribsome.htm
In gel electrophoresis experiment loading dye or buffer is widely used with DNA and RNA to follow the migration of samples. Typical loading buffer composed of two dyes bromophenol blue and xylene cyanol FF. It can be used for both agarose and polyacrylamide gel. Composition of 6x loading dye or buffer:
