Page 39 - IBRO_RNA School_Abstract Book

Virtual Lab Session: Studying the spatio-temporal expres-

sion pattern of lncRNAs

Surendra Singh Patel

CSIR-Institute of Genomics and Integrative Biology,

Mathura Road, Delhi, India

• Collect regenerating earthworm from the bin in 50ml falcon and rinse it

properly in running tap water to remove soil.
• Transfer them into the petri plates with some water to keep the earth-
worms moist, cut the regenerating tissue of earthworm and leave them

into the petri plates to ooze out the soil from alimentary canal, at every 5
min interval wash this regenerated tail from autoclaved MQ water,
repeat this for next 40-45 min (during the process be careful that
earthworms should not die, because they remain alive for at least 45-60
min after cut), if you feel they are about to die then transfer them into

the precooled 4%paraformaldehyde (PFA made in PBS, pH 7.4) for
fixation overnight at 4C.
• Stringent washes with 0.1% tween 20 in PBS (PBST).

• Store them in 100% methanol at 4C for a long time or move ahead
imme-diately to the next step.
• In case of stored worms in methanol prior to in situ hybridization rehy-
drate them with gradients of 90%, 75%,50%,25% & 0.0 % v/v methanol
in PBST for 1hr each.

• Permeabilize earthworms by 20ug/ml Proteinase K for 45-60 min
@55C.
• Fix them again for 15-20 min in a precooled 4% PFA.

• Perform blocking with hybridization buffer only at 65C for 1hour.
• Hybridization using sense and antisense probe @ 65C in hybridization
buffer (50% formamide, 1.3X SSC, 5mM EDTA, 0.2% tween 20, 100ug/
ml heparin, 50ug/ml yeast t-RNA, 5% dextran sulphate in DEPC water)
over-night.

• Washing with

1. 2X SSC, 2min
2. Stringent solution (20% formamide & 0.1X SSC in DEPC treated water) 2
times, 5 min each

3. 0.1X SSC, 3 times, 5 min each
4. 2X SSC, 2 times, 3 min each
5. TBST wash 1-2 times