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
References
  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 guanidinium 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.

'"/>

Source:


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:
*Name:
*Comment:
*Email:
TAG: Total RNA Isolation Protocol

(Date:8/28/2008)...- ViroPharma Incorporated,(...o, president and chief,executive officer of ViroPh...008 Healthcare Conference at 8:00 A.M. ET on Thurs...ld at the Four Seasons Hotel in Boston, MA., Viro...resident,corporate communications of ViroPharma w...
(Date:8/28/2008)...st 28 /- ThromboGenics NV,(...ocused on innovative,treatments for eye disease, v...iness update and its financial results for the six...irst half of 2008, ThromboGenics has achieved a nu...ompany to successfully pursue the,next stage of it...
(Date:8/28/2008)...ewswire-FirstCall/ -- SulphCo, Inc. (the "Company"... with a patented ultrasound,process to desulfurize...d,S. Zeidman to the Company,s Board of Directors.,...,s Board of Directors,",said Dr. Larry D. Ryan, Ch...l benefit from his extensive experience, both in t...
(Date:8/28/2008)...ust 28 /- Strong organic,gr...h product offerings.,The development of Exiqon,s f...rofiling remains on track for year end launch., ...r-earlier period to DKK 28 million, totaling DKK ...Product sales in Q2 2008 increased by 239% on the ...
Breaking Biology Technology:ViroPharma to Present at Three September Healthcare Conferences 2ThromboGenics Announces Half Year Results 2008 2ThromboGenics Announces Half Year Results 2008 3ThromboGenics Announces Half Year Results 2008 4ThromboGenics Announces Half Year Results 2008 5SulphCo Adds New Board Member 2Exiqon A/S: Interim Report for the Period 1 January - 30 June 2008 (Unaudited) 2
...ced today that it is the only Wisconsin member of ...hnology Trainers United Training. United Trainin...ated Information Technology training centers from ...set of standards to be eligible for membership. ... recognized by its peers, customers, and by Micros...
...y rules under the Health Insurance Portability and...y to all health plans, including small plans with ...alth plan? , ,A health plan is any insured or s...ples of health plans include major medical plans, ...ans (EAPs) that provide mental health care, health...
...res about me. He often sends me stock tips. Brittn...uy prescription drugs on the cheap. Octavio? Hes m... seems more interested in enlargement. , ,These ar...line of my e-mail every day, usually arriving over...is cleaning out unsolicited messages from Fidel, E...
...dia the company he founded with his brother Jeremy...ture, this time offering a product for the user si...others, along with Adam Berrey, founded Allaire so...ment tool, ColdFusion. The product soared, as did ...y of 1999. Then, as stocks took their tumble in 20...
Other Biology Technology:Time for health plans to comply with HIPAA privacy rules 2Time for health plans to comply with HIPAA privacy rules 3Putting spam back in the can wont be all that easy 2Onfolio unlocks web research 2Onfolio unlocks web research 3Onfolio unlocks web research 4Onfolio unlocks web research 5
(Date:8/28/2008)...008) A WCS report reveals surprisingly large popul...otected area in Cambodia., , The report counted...0 yellow-cheeked crested gibbons in Cambodia,s Sei...t represents the largest known populations for bot...cted the surveys with the Royal Government of Camb...
(Date:8/28/2008)...ngham have been awarded 2.8 million by the Wellcom...suffering of hundreds of thousands of heart diseas..., In the UK, 2.6 million people suffer from heart...effectively managed with the prescription of beta-...e heart work too hard. , However, a major side e...
(Date:8/27/2008)...searcher Robert Jernigan believes that his researc...t biochemists traditionally believe proteins have ...conducted by Jernigan, director of the L.H. Baker ...s together with Guang Song, an assistant professor..., over a 10-year period shows that not only are pr...
(Date:8/27/2008)...uerzburg will receive a special discretionary priz...n in the Life Sciences. The European Molecular Bio... to a practicing scientist in Europe for outstandi...ward was made in recognition of Tautz,s long-term ... using all available media. , Juergen Tautz is a...
Breaking Biology News(10 mins):Unexpected large monkey population discovered 2New beta-blocker to offer hope to heart and lung sufferers 2Iowa State University researcher shows proteins have controlled motions 2EMBO recognizes German zoologist for public communication 2IH Systems Announces 247 6 million in Financing to Advance RIVA Pharmacy Automation System 5093 1IH Systems Announces 247 6 million in Financing to Advance RIVA Pharmacy Automation System 5093 2October 31 is National Interstitial Cystitis 28IC 29 Awareness Day 5091 1October 31 is National Interstitial Cystitis 28IC 29 Awareness Day 5091 2October 31 is National Interstitial Cystitis 28IC 29 Awareness Day 5091 3October 31 is National Interstitial Cystitis 28IC 29 Awareness Day 5091 4Landmark Report 3A Excess Body Fat Causes Cancer 5089 1Landmark Report 3A Excess Body Fat Causes Cancer 5089 2Landmark Report 3A Excess Body Fat Causes Cancer 5089 3CNS Response Inc Names Henry T Harbin MD to Board of Directors 5087 1CNS Response Inc Names Henry T Harbin MD to Board of Directors 5087 2CNS Response Inc Names Henry T Harbin MD to Board of Directors 5087 3
...cterium in an immune-compromised patient, accordin... The bacterium belongs to the family Acetobactera...nt, some of which are used in industry, such as vi... invasive human disease caused by any of the Aceto...ew bacterium was identified in a patient with chro...
...cterium in an immune-compromised patient, accordin... The bacterium belongs to the family Acetobactera...nt, some of which are used in industry, such as vi... invasive human disease caused by any of the Aceto...ew bacterium was identified in a patient with chro...
...hat may improve construction or repair of bones in...searchers from the American Dental Association Fou...ards and Technology (NIST). , Described in recent...y provides a method for making scaffolds for bone ...n cells and is formed with a cement paste made of ...
...the tail so that they can,t wriggle off in opposit... of the European Molecular Biology Laboratory (EMB... have now discovered that something similar happen... muscle tissue. Their work appears in the current ...e, muscle looks like millions of tiny pistons, sta...
Other Biology News:Soil-bound prions that cause CWD remain infectious 2Scientists discover a new disease-causing bacterium in an immune-compromised patient 2Bioactive cement scaffold may improve bone grafts 2The giant protein titin helps build muscles 2
...ressive centrifuge design and manufacturing, Therm...d Centra-CL3R (refrigerated) general purpose model...,, Quality Control Over Sample Preparation ,...cols and has speed and accuracies to the nearest 5...
p230 trans Golgi Purified Anti-Human clone 15, Isotype Mouse IgG 1 , 50 µg Consult technical datasheet for details.
...) Fragment, Exonuclease Minus, is a DNA-dependent ...´ and the 3´→5´ exonuclease a... I (1,2). It is used for random primer labeling (3...Klenow Fragment, Exonuclease Minus, will leave a s...
...soprostanes produced from arachidonic acid during ...rictor in the rat. 8-iso PGE2 inhibits U-46619 or ...s of 0.5 and 5 µM, respectively. When infus...tion of 4 mg/kg/min, 8-iso PGE2 decreases the GFR ...
Biology Products: