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:10/9/2015)... , Oct. 9, 2015 Governor ... clinical stage biopharmaceutical company focused on developing T-cell receptor ... high-paying jobs with a new expansion project. ... news for the commonwealth as 110 new, high-paying jobs ... Tom Wolf . "My budget proposal includes sustained funding ...
(Date:10/9/2015)...  Pulmatrix, Inc., (NASDAQ: PULM ) will be webcasting ... th Annual BIO Investor Forum on Tuesday, ... --> th Annual BIO Investor Forum ... pm EDT). --> Pulmatrix will be presenting ... on Thursday, October 15, 2015 at 8:30 am EDT. Additionally, ...
(Date:10/8/2015)... BELLINGHAM, Washington, USA, and CARDIFF, UK (PRWEB) , ... October 08, ... ... noninvasive light-based imaging technology to literally see inside the living brain, providing a new ... over time. , The work is reported by Woo June Choi and Ruikang Wang ...
(Date:10/8/2015)...  Genetic testing for mutations beyond those currently recommended ... couples at risk of having a child with cystic ... 2015 American Society of Human Genetics (ASHG) Annual Meeting, ... Maryland . The study is one of four ... are presenting at the conference this week demonstrating the ...
Breaking Biology Technology:
... Commercialization and Supply Agreement, Company to Host Conference Call at 12:30 p.m. ... ... 2007, LEXINGTON, Mass., Sept. 19 Indevus,Pharmaceuticals, Inc. (Nasdaq: ... acquisition of Esprit Pharma, has obtained the,United States rights to SANCTURA(R) and ...
... Ventures, Kleiner Perkins ... Ventures, Strong Endorsement of Technology Pioneer at Forefront of Developing No- ... Compromise Biofuels, ... renewable hydrocarbon biofuels, today,announced that it closed the first tranche of its $70 ...
... - IDF Launches New Guideline for the ... the new global guideline for diabetes care which,includes ... people with diabetes should have their blood glucose ... diabetes control and reduce the,risk of complications, particularly ...
Cached Biology Technology:
(Date:9/24/2015)... September 24, 2015 ... 2015 Kerv ( ... lanserar idag världens första kontaktlösa betalningsring på ... 77 000 GBP för massproduktion via crowdfunding.  ... ) , Kerv-bärare kan göra ...
(Date:9/10/2015)... , Sept. 10, 2015 Pursuant Health ... Wellness to create an interactive, image-based health risk ... and wellness kiosk.  The unique assessment quantifies user ... number that suggests an individual,s biological age based ... as measured by the kiosk. Comprised ...
(Date:9/8/2015)... BOSTON , Sept. 8, 2015  Affectiva, ... of Emotion as a Service, a new data ... Based on Affectiva,s highly accurate and ... and analytics capabilities, slated to transform industries such ... app development, retail, customer experience, online education, HR, ...
Breaking Biology News(10 mins):
... For the first time, researchers have been able to confine ... role in photosynthesis, without having to pin it down so ... first practical application to proteins of a recently developed technique, ... the dynamic behavior of the molecule for more than one ...
... system science disciplines recently gathered to address a major ... future? Of course, possible answers to this question ... conditions were to alter local vegetation, how would this ... questions with certainty would allow us to manage better ...
... , SEATTLE, WA March 10, 2010 -- The ... genome sequences of a human family of four. The findings ... the University of Luxembourg was published online today by Science ... benefit of sequencing entire families, including lowering error rates, identifying ...
Cached Biology News:
Request Info...
Recombinant Equine TNF-alpha/TNFSF1A, CF...
Recombinant Equine IL-4, CF...
actin from rabbit muscle Cell Biology Cytoskeletal Probes Actin and Actin Probes...
Biology Products: