Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 234

2 m L of 0.1 M DTT, and 1 m L of RNasin (Promega). Incubate at 42 C for 2 min
before adding 1 m L of Superscript III Reverse Transcriptase enzyme (Invitrogen).
Incubate at 42 C for 50 min and 70 C for 15 min. Add 1 m L of RNaseH (Invitrogen),
and incubate at 37 C for 30 min. Dilute an aliquot of the cDNA reaction 1:5 for use in
qPCR. Analyze samples for the primary or processed miRNA or mRNA of interest
by PCR or qPCR as described above in Section II.
M. Gel Filtration Analysis of miRNPs
Gel filtration has been used to fractionate cell extracts from C. elegans,
Drosophila, and human cells to determine the sizes of complexes corresponding
to RNAi activity, siRNAs, miRNAs, or miRNP/RISC ( Caudy et al., 2002, 2003;
Chan et al., 2008; Gregory et al., 2005; Pham et al., 2004 ). Gel filtration can also be
used to detect cofractionation of proteins of interest with miRNAs or to purify
fractions that possess miRNA-related activities, that is miRNA binding or pre-
miRNA processing.
1. Cell Extract Preparation
Harvest mixed-staged (or staged) worms grown in S medium supplied with E. coli
HB101. Clean the worms by sucrose floatation and then wash three times with 0.1 M
NaCl. Store the worms at 80 C. Thaw 1 mL of packed worms on ice and wash
twice with wash buffer (50 mM Tris-HCl pH 7.5 and 10 mM potassium acetate).
Resuspend the worms in 4 mL of homogenization buffer [50 mM Tris-HCl pH 7.5,
10 mM potassium acetate, 5 mM DTT, 10 U/mL SuperaseIn RNase inhibitor
(Ambion), 10% glycerol, and 1 Roche Complete protease inhibitor, EDTA-free]
and incubate on ice for 20 min with intermittent agitation. Transfer the worms to a
7 mL glass dounce homogenizer tube (Kontes Glass Co., Vineland, N.J., article no.
885303-0007) and homogenize by 30 strokes on ice using the tight-fitting pestle B
(Kontes Glass Co., Vineland, N.J., article no. 885302-0007). Transfer homogenate to
a 15-mL tube and add magnesium acetate to 2 mM and adjust the concentration of
potassium acetate to 100 mM. Incubate the homogenate on ice for 20 min with
intermittent agitation and then aliquot the homogenate to 1.5 mL eppendorf tubes.
Centrifuge the tubes at 16,100 gat4 C for 30 min. Store the supernatant at 80 C.
2. Gel Filtration
Equilibrate a Superdex-200 HR 10/30 column with cold homogenization buffer.
Load 400 m L of cell extract into the column. Run the chromatography at 4 C at the
rate of 0.35 mL per minute using AKTA FPLC (Pharmacia) and collect 60 fractions
(0.5 Ll each). Use the following size markers: thyroglobulin (669 kDa), ferritin
(440 kDa), catalase (232 kDa), aldolase (158 kDa), and albumin (67 kDa). For
monitoring miRNAs in the fractions, mix 250 m L of fraction sample with 750 m L