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Need help on RNA preparation for RNA sequencing

RIN value above 9

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7 replies to this topic

#1 Trys

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Posted 18 December 2013 - 05:27 PM

Hi Friends,

 

I want to prepare RNA for RNA sequencing to establish library.  So far my RNA prep's RIN value is only ~7.6 to 8.1 using Trizol reagents. I really need help to improve my RNA's RIN value above 9 by either improving Trizol protocol or other methods.

 

Thank you in advance!!!

 

Trys



#2 memari

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Posted 19 December 2013 - 12:31 PM

You can clean your RNA by a column base kit.

Like this one which is very cheap and as good as RNeasy Plus Mini kit of Qiagen. That is it has two column. One for removing DNA and particles, and the second column which is for RNA binding.

 

Blood/Cultured Cell Total RNA Mini Kit (300prep)   $363.00  CDN from Favorgen Biotech Corporation

https://cedarlanelab...tail/FABRK001-2

 

http://www.favorgen....extraction.html

 

Copare the price:

Qiagen RNeasy Plus Mini Kit (250 prep$1,628.00

Favorgen Blood/Cultured Cell Total RNA Mini Kit (300 prep)   $363.00

 

and a tips and triks for using all column base purification kits:

use 14000 RPM (20800 rcf) for all steps for anything from purification of DNA, RNA and etc..


Edited by memari, 19 December 2013 - 12:33 PM.

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#3 Trys

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Posted 19 December 2013 - 06:08 PM

Hi Memari,

 

Thank you so much for your inputs!! So, it seems Trizol has limits for RNA with RIN>9.   I guess that column based approaches can readily reach this levels.

Thank you again and I will try it,

 

Trys



#4 memari

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Posted 19 December 2013 - 06:43 PM

you can clean this RNA that you have obtained from Trizol method.

 

Just add lysis buffer of column base kit to this RNA and go forward.


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Babak Memari

#5 Trys

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Posted 22 December 2013 - 11:51 AM

Hi Babak,

 

I isolated some RNA by Qiagen kit and also purified some of my previous RNAs, the RIN did improve. But I felt that the yield from column kit is lower than TRIZOL, so is it a better that, during RNA isolation, use Trizol first and then purify further by column?  Since I have small amount of materials, yield is also kind of important to me,

When you have time, could you advise me further?

Thank you so much again,

 

Trys



#6 memari

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Posted 23 December 2013 - 08:41 AM

https://www.youtube....I-lH-mgvI#t=626

 

http://babakmemari.w...delinefor-qpcr/


Edited by memari, 23 December 2013 - 08:42 AM.

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#7 Mohamed 1984

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Posted 26 February 2014 - 11:45 AM

One suggestion is to make on column DNAse digestion. The yield of RNA will be somehow reduced but might be helpful to raise your RIN



#8 Mohamed 1984

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Posted 26 February 2014 - 11:46 AM

By the way, Is any one know if these two column kits is available for miRNAs or not ? 






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