Rna micro kit

Transgenic mice and FVB/NJ breeders were obtained from Jackson Laboratories (stock # 005058 and 001800). The resultant progeny (Tg SMN2 2Hung^tg/O; Smn1^TmHung/WT) were intercrossed to produce “experimental” breeders (Tg SMN2 2Hung^O/O; Smn1 ^TmHung/WT). These were then mated with 005058 animals to produce litters which are 50% healthy (Tg SMN2 2Hung^tg/O; Smn1^TmHung/WT-) and 50% SMA (Tg SMN2 2Hung^tg/O; Smn1^{TmHung/ TmHung}). Animals were maintained in accordance with international guidelines with approval from the Indiana University School of Medicine Animal Care and Use Committee (IUSM IACUC) as described in protocol #10646. For spinal cord removal, pups were euthanized by decapitation at post-natal day 9 and total RNA was harvested as directed using the Trizol method detailed in the RNA micro kit (Invitrogen, # 12183016)

The procedure for the mRNA assay was modified from a previous study62 (link). Total RNA was extracted from tissue punches of the PrL and IL using an RNA Micro kit (Invitrogen). mRNA was reverse-transcribed into cDNA using the First Strand cDNA Synthesis kit (Invitrogen). For the amplification of PKMζ cDNA, the sequences of specific primers were the following: forward (5′-CCTTCTATTAGATGCCTGCTCTCC-3′) and reverse (5′-TGAAGGAAGGTCTACACCATCGTTC-3′)18 (link). Complementary DNA amplification was performed in a CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA). The samples were normalized to β-tubulin-4, and the comparative cycle threshold method was used to calculate differences in gene expression between samples63 (link). Message RNA status in different manipulation groups is presented as fold changes relative to the naive group.