RNA Quality Control

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(→RNA Image galleries: added example mouse total RNA electropherograms)
 
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*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.
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==Checking RNA/cRNA samples on an agilent bioanalyzer==
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==Checking RNA/cRNA samples on an Agilent [[Bioanalyzer]]==
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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.
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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.
   
   
===RNA Image galleries===
===RNA Image galleries===
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<div style="padding: 10px; width: 720px; border: 2px solid #000000;">
The following are examples of typical Human RNA examined on an Agilent 2100 BioAnalyser.
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.
RNA electropherograms have different profiles depending on species, tissue and starting material quality.
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<gallery>
<gallery>
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Image:HumanRNA.png
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Image:Human RNA good quality.jpg|Human RNA good quality
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Image:Image name|thumb|Human
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Image:Mouse total RNA undegraded.PNG|Mouse total RNA undegraded, RIN 10
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Image:Image name|thumb|Human slightly degraded
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Image:Mouse total RNA slightly degraded.PNG|Mouse total RNA slightly degraded, RIN 9.4
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Image:Image name|thumb|Human very degraded
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Image:Arabidopsis RNA good quality.jpg|Arabidopsis RNA good quality
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Image:Image name|thumb|Arabidopsis
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Image:Arabidopsis single cell aRNA.jpg|Arabidopsis single cell aRNA
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Image:Image name|thumb|Human brain
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Image:Barley RNA good quality.jpg|Barley RNA good quality
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Image:Image name|thumb|Human muscle
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Image:Wheat RNA good quality.jpg|Wheat RNA good quality
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Image:Image name|thumb|Human blood
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Image:Image name|thumb|Drosphila
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Image:Image name|thumb|Drosphila degraded
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Image:Image name|thumb|Arabidopsis
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Image:Image name|thumb|Arabidopsis degraded
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</gallery>
</gallery>
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Explanation of electropherograms.
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</div>
===cRNA Image galleries===
===cRNA Image galleries===
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<div style="padding: 10px; width: 720px; border: 2px solid #000000;">
The following are examples of typical cRNA products examined on an Agilent 2100 BioAnalyser.
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.
cRNA electropherograms have different profiles depending on species and starting material quality.
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<gallery>
<gallery>
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Image:Image name|thumb|Human cRNA 10ug starting RNA
 
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Image:Image name|thumb|Human cRNA 5ug starting RNA
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Image:Affymetrix One cycle Human cRNA good quality.jpg|Affymetrix One cycle Human cRNA good quality
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Image:Image name|thumb|Human cRNA 1ug starting RNA
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Image:Affymetrix Two cycle Human cRNA good quality.jpg|Affymetrix Two cycle Human cRNA good quality
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Image:Image name|thumb|Human cRNA 100ng starting RNA
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Image:Affymetrix one cycle Arabidopsis cRNA good quality.jpg|Affymetrix one cycle Arabidopsis cRNA good quality
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Image:Image name|thumb|Human cRNA 10ng starting RNA
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Image:Affymetrix one cycle Arabidopsis single cell aRNA cRNA.jpg|Affymetrix one cycle Arabidopsis single cell aRNA cRNA
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Image:Affymetrix one cycle Barley cRNA good quality.jpg|Affymetrix one cycle Barley cRNA good quality
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Image:Affymetrix one cycle Wheat cRNA good quality.jpg|Affymetrix one cycle Wheat cRNA good quality
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Image:Affymetrix one cycle Wheat cRNA poor quality.jpg|Affymetrix one cycle Wheat cRNA poor quality
</gallery>
</gallery>
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Explanation of electropherograms.
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</div>
==Checking RNA/cRNA Samples on a denaturing formaldehyde gel==
==Checking RNA/cRNA Samples on a denaturing formaldehyde gel==
I still need to add a formaldhyde gel protocol, anyone else got one?
I still need to add a formaldhyde gel protocol, anyone else got one?
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==Contact==
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*mail to jamesdothadfieldatcancerdotorgdotuk
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*This page was created by James Hadfield on 12 October 2006. I hope you found it useful.
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[[Category:Protocol]]
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[[Category:In vitro]]
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[[Category:RNA]]

Current revision

Contents

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.

Human RNA good quality

Mouse total RNA undegraded, RIN 10

Mouse total RNA slightly degraded, RIN 9.4

Arabidopsis RNA good quality

Arabidopsis single cell aRNA

Barley RNA good quality

Wheat RNA good 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.

Affymetrix One cycle Human cRNA good quality

Affymetrix Two cycle Human cRNA good quality

Affymetrix one cycle Arabidopsis cRNA good quality

Affymetrix one cycle Arabidopsis single cell aRNA cRNA

Affymetrix one cycle Barley cRNA good quality

Affymetrix one cycle Wheat cRNA good quality

Affymetrix one cycle Wheat cRNA poor 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.
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