Tri reagent

Placentas from the contralateral horn were bisected, and one half was
stored in TriReagent (Sigma) at -80°C. RNA was isolated from seven
SMNΔ7 placentas and seven littermate controls (5
SMN^+/+; 2 SMN^+/-) The tissue
was homogenized in TriReagent using an OMNI International GLH1 rotor, and total
RNA extracted according to manufacturer's protocol with modification, as
follows: after chloroform extraction, the aqueous phase was further purified by
using the RNeasy mini kit (Qiagen), according to manufacturer's
protocol. Two micrograms of total RNA were reverse transcribed using Invitrogen
superscript III reverse transcriptase kit and random hexamers to generate first
strand cDNA as outlined in manufacturer's protocol.
Forward and reverse primers and probes were synthesized by Integrated DNA
Technologies (Table 1). Real-time PCR was
performed in an ABI PRISM 7500 real time detection system using SABiosciences
RT² Sybr Green/ROX qPCR master mix or Applied Biosystems Taqman
Universal PCR master mix on settings 50°C for 2mins, 95°C for
10mins, and 40 cycles of 95°C for 15 secs, 60° C for 1min. All
reactions were performed in triplicate. Dissociation curves were examined to
ensure amplification of a single product. Reaction efficiency was validated by
using serial dilutions of template cDNA. Relative levels of mRNA were calculated
using the ΔΔC_t method, with normalization to 18s gene
expression[18 (link)].

Tissues were snap-frozen in liquid nitrogen immediately after dissection and stored at −80°C until used. For RNA extraction, the tissues were soaked in TRI-reagent (Sigma) and were homogenized in Bead Ruptor24e (OMNI) with stainless steel beads, and then proceeded by a standard TRI-reagent based RNA extraction protocol. RNA concentration was determined using NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific). RNA quality was validated using 2200 TapeStation (Agilent).