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Old 03-26-2010, 08:06 AM   #1
rodr08
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Default Rationale for Superscript II RT use in Illumina 1st strand synthesis


Hello party people,
Does anyone know why Illumina's 1st strand synthesis protocol (for RNA-seq) uses Superscript II (SSII, Invitrogen) as opposed to Superscript III (SSIII)?

Besides it lower thermal stability and shorter half life, SSII exhibits tailing activity, adding "2 to 3 bases in a template independent fashion to the end of a primer." (taken from invitrogen product notes)

I am a novice, still learning about the seq technology and illumina protocols so bear with me. Shouldn't introducing these extra bases effectively decrease the length of your final sequence read?
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Old 03-30-2010, 08:21 AM   #2
rodr08
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I spoke with Illumina technical support and got the answer to my question and some additional information that might be helpful to others.

They developed their protocols using SSII and never tested it with SSIII. The representative called this move "Institutional Inertia." The rep went on to say customers had reported using SSIII successfully for mRNA-seq and sRNA V1.5 library preps.
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Old 05-02-2010, 02:04 AM   #3
peromhc
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Quote:
Originally Posted by rodr08 View Post
They developed their protocols using SSII and never tested it with SSIII. The representative called this move "Institutional Inertia." The rep went on to say customers had reported using SSIII successfully for mRNA-seq and sRNA V1.5 library preps.
Interestingly, If you look at the mRNA prep protocol revision A (not the current revision D) SSIII is used... I had wondered if it had something to do with the lower optimal temp of SSII (42 degrees) versus 50 for SSIII

I recently used SSIII for a mRNA prep without a problem. While Illumina may not officially support it, it certainly works.
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Old 05-03-2010, 11:36 AM   #4
rodr08
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Thanks for sharing this information
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