RNA Quality Control: Difference between revisions

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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.


==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.
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===
<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.
<gallery>
<gallery>
Image:HumanRNA.png
Image:Human RNA good quality.jpg|Human RNA good quality
Image:Image name|thumb|Human
Image:Mouse total RNA undegraded.PNG|Mouse total RNA undegraded, RIN 10
Image:Image name|thumb|Human slightly degraded
Image:Mouse total RNA slightly degraded.PNG|Mouse total RNA slightly degraded, RIN 9.4
Image:Image name|thumb|Human very degraded
Image:Arabidopsis RNA good quality.jpg|Arabidopsis RNA good quality
Image:Image name|thumb|Arabidopsis
Image:Arabidopsis single cell aRNA.jpg|Arabidopsis single cell aRNA
Image:Image name|thumb|Human brain
Image:Barley RNA good quality.jpg|Barley RNA good quality
Image:Image name|thumb|Human muscle
Image:Wheat RNA good quality.jpg|Wheat RNA good quality
Image:Image name|thumb|Human blood
Image:Image name|thumb|Drosphila
Image:Image name|thumb|Drosphila degraded
Image:Image name|thumb|Arabidopsis
Image:Image name|thumb|Arabidopsis degraded
</gallery>
</gallery>
Explanation of electropherograms.
</div>


===cRNA Image galleries===
===cRNA Image galleries===
<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.
<gallery>
<gallery>
Image:Image name|thumb|Human cRNA 10ug starting RNA


Image:Image name|thumb|Human cRNA 5ug starting RNA
Image:Affymetrix One cycle Human cRNA good quality.jpg|Affymetrix One cycle Human cRNA good quality
Image:Image name|thumb|Human cRNA 1ug starting RNA
Image:Affymetrix Two cycle Human cRNA good quality.jpg|Affymetrix Two cycle Human cRNA good quality
Image:Image name|thumb|Human cRNA 100ng starting RNA
Image:Affymetrix one cycle Arabidopsis cRNA good quality.jpg|Affymetrix one cycle Arabidopsis cRNA good quality
Image:Image name|thumb|Human cRNA 10ng starting RNA
Image:Affymetrix one cycle Arabidopsis single cell aRNA cRNA.jpg|Affymetrix one cycle Arabidopsis single cell aRNA cRNA
Image:Affymetrix one cycle Barley cRNA good quality.jpg|Affymetrix one cycle Barley 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
</gallery>
</gallery>
Explanation of electropherograms.
</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?
==Contact==
*mail to jamesdothadfieldatcancerdotorgdotuk
*This page was created by James Hadfield on 12 October 2006. I hope you found it useful.
[[Category:Protocol]]
[[Category:In vitro]]
[[Category:RNA]]

Latest revision as of 02:52, 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.

  • Human RNA good quality

    Human RNA good quality

  • Mouse total RNA undegraded, RIN 10

    Mouse total RNA undegraded, RIN 10

  • Mouse total RNA slightly degraded, RIN 9.4

    Mouse total RNA slightly degraded, RIN 9.4

  • Arabidopsis RNA good quality

    Arabidopsis RNA good quality

  • Arabidopsis single cell aRNA

    Arabidopsis single cell aRNA

  • Barley RNA good quality

    Barley RNA good quality

  • Wheat 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 One cycle Human cRNA good quality

  • Affymetrix Two cycle Human cRNA good quality

    Affymetrix Two cycle Human cRNA good quality

  • Affymetrix one cycle Arabidopsis cRNA good quality

    Affymetrix one cycle Arabidopsis cRNA good quality

  • Affymetrix one cycle Arabidopsis single cell aRNA cRNA

    Affymetrix one cycle Arabidopsis single cell aRNA cRNA

  • Affymetrix one cycle Barley cRNA good quality

    Affymetrix one cycle Barley cRNA good quality

  • Affymetrix one cycle Wheat cRNA good quality

    Affymetrix one cycle Wheat cRNA good quality

  • Affymetrix one cycle Wheat cRNA poor 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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