User:Kristina M Holton/Ovarian Notebook 2007 7 2

miRNA Extraction using 1st protocol and Qiagen miRNEasy Kit

• Large Fibroma -> very very fatty
• Used 3ml TRIzol from Invitrogen
• While standing, TRIzol and fat start to pellet at the bottom; removed supernatants and centrifuged both pellet and supernatant
• After centrifugation, already appeared to be phase separation
• Still added glycogen + chloroform, stand RT for 10min then centrifuge 10000g 20min
• Added EtOH: large amount of white, "SDS-like" precipitate
• Very difficult to add precipitate to RNeasy Mini spin columns, even with additional EtOH

Differences in 2nd protocol (probable fixes):

• Use 2ml QIAzol for lysis

If: no debris accumulates and the QIAzol remains pink

• • Skip first centrifugation step + removing the supernatant

Else: (Brown QIAzol or debris accumulation)

• • Centrifuge and remove supernatant
• Try to add all precipitate from EtOH step