Difference between revisions of "RNA Quality Control"

From OpenWetWare
Jump to: navigation, search
(RNA Image galleries: added example mouse total RNA electropherograms)
 
(2 intermediate revisions by the same user not shown)
Line 9: Line 9:
 
*For Affymetrix labeling reactiosns; concentrations of cRNA are generally around 1000-1500ng/µl for a 40µl IVT reaction using 20µl of cDNA template.
 
*For Affymetrix labeling reactiosns; concentrations of cRNA are generally around 1000-1500ng/µl for a 40µl IVT reaction using 20µl of cDNA template.
  
==Checking RNA/cRNA samples on an agilent bioanalyzer==
+
==Checking RNA/cRNA samples on an Agilent [[Bioanalyzer]]==
 
Agilent  [http://www.chem.agilent.com/Scripts/PDS.asp?lPage=51 BioAnalyzer] Gel electrophoresis of the RNA is done to estimate the quality of the RNA. The bioanalyzer software produces a RIN (RNA integrity number) this gives a measure of sample quality that is not subjective. Howver it needs to be used with some caution and is not as useful as it sounds.
 
Agilent  [http://www.chem.agilent.com/Scripts/PDS.asp?lPage=51 BioAnalyzer] Gel electrophoresis of the RNA is done to estimate the quality of the RNA. The bioanalyzer software produces a RIN (RNA integrity number) this gives a measure of sample quality that is not subjective. Howver it needs to be used with some caution and is not as useful as it sounds.
 
   
 
   
Line 18: Line 18:
 
<gallery>
 
<gallery>
 
Image:Human RNA good quality.jpg|Human RNA good quality
 
Image:Human RNA good quality.jpg|Human RNA good quality
Image:Human RNA slightly degraded.jpg|Human RNA slightly degraded
+
Image:Mouse total RNA undegraded.PNG|Mouse total RNA undegraded, RIN 10
Image:Human RNA very degraded.jpg|Human RNA very degraded
+
Image:Mouse total RNA slightly degraded.PNG|Mouse total RNA slightly degraded, RIN 9.4
Image:Human RNA brain.jpg|Human RNA brain
 
Image:Human RNA muscle.jpg|Human RNA muscle
 
Image:Human RNA blood.jpg|Human RNA blood
 
Image:Drosphila RNA good quality.jpg|Drosphila RNA good quality
 
 
Image:Arabidopsis RNA good quality.jpg|Arabidopsis RNA good quality
 
Image:Arabidopsis RNA good quality.jpg|Arabidopsis RNA good quality
Image:Arabidopsis RNA slightly degraded.jpg|Arabidopsis RNA slightly degraded
 
 
Image:Arabidopsis single cell aRNA.jpg|Arabidopsis single cell aRNA
 
Image:Arabidopsis single cell aRNA.jpg|Arabidopsis single cell aRNA
 
Image:Barley RNA good quality.jpg|Barley RNA good quality
 
Image:Barley RNA good quality.jpg|Barley RNA good quality
 
Image:Wheat RNA good quality.jpg|Wheat RNA good quality
 
Image:Wheat RNA good quality.jpg|Wheat RNA good quality
Explanation of electropherograms.
 
 
</gallery>
 
</gallery>
 
</div>
 
</div>
Line 46: Line 40:
 
Image:Affymetrix one cycle Wheat cRNA good quality.jpg|Affymetrix one cycle Wheat cRNA good quality
 
Image:Affymetrix one cycle Wheat cRNA good quality.jpg|Affymetrix one cycle Wheat cRNA good quality
 
Image:Affymetrix one cycle Wheat cRNA poor quality.jpg|Affymetrix one cycle Wheat cRNA poor quality
 
Image:Affymetrix one cycle Wheat cRNA poor quality.jpg|Affymetrix one cycle Wheat cRNA poor quality
Image:Affymetrix Two cycle Human cRNA 100ng starting.jpg|Affymetrix Two cycle Human cRNA 100ng starting
 
Image:Affymetrix Two cycle Human cRNA 10ng starting.jpg|Affymetrix Two cycle Human cRNA 10ng starting
 
Image:Affymetrix Two cycle Human cRNA 5ng starting.jpg|Affymetrix Two cycle Human cRNA 5ng starting
 
Image:Affymetrix Two cycle Human cRNA 1ng starting.jpg|Affymetrix Two cycle Human cRNA 1ng starting<br>
 
Explanation of electropherograms.
 
 
</gallery>
 
</gallery>
 
</div>
 
</div>

Latest revision as of 01:52, 5 May 2011

Quality control of RNA

RNA Quality control This protocol will assist you in performing RNA quality control for a variet of purposes; Microarray, real-time and Northern analysis.

Checking RNA/cRNA samples on a nanodrop Spec

  • Check concentration, A260/A280, A260/A230 on Nanodrop Spectrophotometer.
  • Maintain the A260/A280 ratio close to 2.0 for pure RNA (ratios between 1.9 and 2.1 are acceptable).
  • For Affymetrix labeling reactiosns; concentrations of cRNA are generally around 1000-1500ng/µl for a 40µl IVT reaction using 20µl of cDNA template.

Checking RNA/cRNA samples on an Agilent Bioanalyzer

Agilent BioAnalyzer Gel electrophoresis of the RNA is done to estimate the quality of the RNA. The bioanalyzer software produces a RIN (RNA integrity number) this gives a measure of sample quality that is not subjective. Howver it needs to be used with some caution and is not as useful as it sounds.

RNA Image galleries

The following are examples of typical Human RNA examined on an Agilent 2100 BioAnalyser. RNA electropherograms have different profiles depending on species, tissue and starting material quality.

cRNA Image galleries

The following are examples of typical cRNA products examined on an Agilent 2100 BioAnalyser. cRNA electropherograms have different profiles depending on species and starting material quality.

Checking RNA/cRNA Samples on a denaturing formaldehyde gel

I still need to add a formaldhyde gel protocol, anyone else got one?

Contact

  • mail to jamesdothadfieldatcancerdotorgdotuk
  • This page was created by James Hadfield on 12 October 2006. I hope you found it useful.