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Revision as of 12:19, 5 July 2012

Micro-array: RNA isolation and cDNA synthesis

Isolated RNA from the harvested cells (Badmeat/Freshmeat 02/07 & 03/07)using:

• Macaloïd method (see protocol File:Groningen RR20120705 RNAisolation.pdf)
• Roche High pure RNA isolotion (see protocol here

Quality check with Agilent bio-analyser (protocol.pdf and nanodrop (protocol

Results nanodrop:

• Fresh meat - 02/07: 4458,6 ng/ul; 260/280 = 1,09
• Bad meat - 02/07: 3667,0 ng/ul; 260/280 = 1,97
• Fresh meat - 03/07: 4353,9 ng/ul; 260/280 = 1,54
• Bad meat - 03/07: 3630,2 ng/ul; 260/280 = 1,96

cDNA synthesis (in duplo (named a/b in the rest of the notebook)) using the following mixture:

• Fresh meat - 02/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers
• Bad meat - 02/07: 4,1 ul sample; 11,9 ul H20; 2 ul random nonamers
• Fresh meat - 03/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers
• Bad meat - 03/07: 4,2 ul sample; 11,8 ul H20; 2 ul random nonamers

• mixed, 5' 70 degrees Celsius.
• 10 min RT
• on ice

• Further with Reverse transcription using the Superscript III reverse transcriptase kit (File:Groningen RR20120705 cDNAprotocol.pdf)
• 18 hours, 42 degrees Celsius (O/N)