Navigation Links
Total RNA Isolation Protocol 1,2,3

  1. Sample Preparation:
    1. To extract RNA from washed and pelleted cultured cells, add 200 l 4 M Guanidinium Isothiocyanate Solution (4M Guanidinium Isothiocyanate, 25 mM Sodium Citrate, pH 7.0, 1 M b-Mercaptoethanol) to 0.5 x 104 cells - 1 x 106 cells.
    2. To directly extract RNA from cultured cells growing in monolayer, add 200 l 4 M Guanidinium Isothiocyanate Solution directly to each well of a 6, 12 or 24 well plate. Add 100 l of the 4 M Guanidinium Isothiocyanate Solution directly to each well of a 48 or 96 well plate.
  2. Homogenize monolayer cells by pipetting the mixture up and down several times, taking care to "wash" cell material free from the culture dish, tube or well in the process. Homogenize washed, pelleted cells by pipetting the mixture up and down until the pellet is fully suspended. Use a small bore pipet to collect the cell homogenate.
  3. Transfer all of the homogenate to a pre-spun (12,00016,000 x g for 12 minutes) PLG 2 ml Heavy tube.
  4. Add 20 l (10 l per sample for 48 or 96 well plates) 2.0 M Sodium Acetate, pH 4.0 to the sample, cap the PLG tube and mix briefly.
  5. Add 200 l (100 l per sample for 48 or 96 well plates) water-saturated Phenol to the sample, cap the PLG tube, and mix thoroughly by repeated inversion. Do not vortex.
  6. Add 60 l (30 l per sample for 48 or 96 well plates) Chloroform: Isoamyl Alcohol (CI, 49:1) to the sample in the same PLG tube and mix thoroughly by repeated gentle inversion. Do not vortex.
  7. Incubate on ice for 10 minutes.
  8. Centrifuge at 12,00016,000 x g for 5 minutes in a microcentrifuge to separate the phases.
  9. Add 200 l (100 l per sample from 48 or 96 well plates) Phenol-Chloroform-Isoamyl Alcohol (PCI, 50:49:1) to the aqueous phase in the same PLG tube. Mix thoroughly by repeated gentle inversion. Do not vortex.
  10. Centrifuge at 12,00016,000 x g for 5 minutes to separate the phases.
  11. Collect resultant aqueous phase to an RNase-free microcentrifuge tube, add an equal volume of 100% Isopropanol, and mix by repeated inversion.
  12. Centrifuge at 12,00016,000 x g for 20 minutes.
  13. Discard resultant supernatant and wash pellet several times with 200 l 70% Ethanol, using 23 minutes at 12,00016,000 x g to re-pellet the sample.
    Note: Samples may be stored in the 70% Ethanol wash at this stage at 70C or colder for extended periods.
  14. Discard final wash and dry pellet at room temperature.
  15. Re-dissolve pellet in a suitable volume (510 l) of RNase-free water. Store the RNA solution at 70C.
    Note: Absorbance determinations should be performed in RNase-free TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0).15
  1. Chirgwin, J.M. et al. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem. 18:5294-5299.
  2. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guan idinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159.
  3. Birnboim, H.C. (1988) Rapid extraction of high molecular weight RNA from cultured cells and granulocytes for Northern analysis. Nucl. Acids Res. 16:1487-1497.
  4. Wilfinger, W.W. et al. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22:474-481.



Page: All 1 2 3

Related biology technology :

1. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
2. High-Throughput Isolation of Total RNA
3. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture Microdissection
4. Fast Isolation of Total RNA from Small Numbers of Cells
5. Isolate It All: siRNA miRNA Total RNA Native Protein
6. Isolate Total RNA and Protein From the Same Sample
7. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
8. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
9. Total RNA from Whole Blood for Expression Profiling
10. RNAPrep - Total RNA Isolation Purification
11. Total RNA Isolation From 500 l Body Fluid
Post Your Comments:

(Date:12/1/2015)... 2015 Researchers at the Broad Institute of ... Research at MIT have engineered changes to the revolutionary ... "off-target" editing errors. The refined technique addresses one of ... editing. Science , Feng Zhang ... approximately 1,400 amino acids that make up the Cas9 ...
(Date:12/1/2015)... , Dec. 1, 2015  The Minnesota High ... of the 2015 Tekne Award in the Small and ... at the Minneapolis Convention Center, ... played a significant role in developing new technologies that ... around the world. Clostridium difficile infection ...
(Date:12/1/2015)... Frederick, MD (PRWEB) , ... December 01, 2015 ... ... management solutions provider, announces that its best selling system laboratory animal colony management ... ezColony® Cloud today, without investing in on-site IT resources., , ...
(Date:12/1/2015)... Dr. Harry Lander , President of Regen, expands his role to ... and recruits five distinguished scientists to join ... expands his role to include serving as ... scientists to join advisory team --> Dr. Harry ... serving as Chief Science Officer and ...
Breaking Biology Technology:
(Date:10/29/2015)... , Oct. 29, 2015  Rubicon Genomics, Inc., ... U.S. distribution of its DNA library preparation products, ... Rubicon,s new ThruPLEX Plasma-seq kit. ThruPLEX Plasma-seq has ... preparation of NGS libraries for liquid biopsies--the analysis ... and prognostic applications in cancer and other conditions. ...
(Date:10/29/2015)... , Oct. 29, 2015 Today, ... a partnership with 2XU, a global leader in ... a smart hat with advanced bio-sensing technology. The ... athletes to monitor key biometrics to improve overall ... partnership, the two companies will bring together the most ...
(Date:10/27/2015)... 2015 Synaptics Inc. (NASDAQ: SYNA ), the ... has adopted the Synaptics ® ClearPad ® ... its newest flagship smartphones, the Nexus 5X by LG ... --> --> Synaptics works closely ... collaboration in the joint development of next generation technologies. ...
Breaking Biology News(10 mins):