Abi prism 7900 sequence detector

To analyze GSC miRNA expression, total RNA was prepared using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA was labeled and hybridized to the Agilent‐019118 array following the manufacturer's instructions. Microarray analysis was performed as previously described (Felli et al., 2010).
For real‐time PCR, 50 ng of RNA was reverse‐transcribed with TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). Real‐time PCR for miR‐23a‐3p (miRBase ID MIMAT0000078), miR‐27a‐3p (miRBase ID MIMAT0000084), and miR‐9‐3p (miRBase ID MIMAT0000442) was performed using TaqMan^® MicroRNA Assays protocol (assay ID 000399, ID 000408, ID 002231; Applied Biosystems). All reactions were run in duplicate. Normalization was performed by using RNU6B primer kit (ID 001093; Applied Biosystems). RT‐PCR was performed using an ABI Prism 7900 Sequence Detector (Applied Biosystems).

Total RNAs were isolated with the RNeasy mini kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. Two micrograms of total RNA was transcribed into complementary DNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA). Quantification of mRNA levels of the following genes was done using TaqMan gene expression assays (Applied Biosystems) and an ABI Prism 7900 sequence detector (Applied Biosystems): PMAIP1 (#Hs00560402_m1), BCL2L1 (#Hs00236329_m1), BAK1 (#Hs00832846_g1), BBC3 (#Hs00248075_m1), MCL1 (#Hs01050896_m1), BCL2L11 (#Hs01076940_m1), BID (#Hs00609632_m1), BAX (#Hs00180269_m1), TP53 (#Hs01034249_m1). qRT-PCR reactions were performed in triplicates for each sample and were normalized to the expression of the housekeeping gene HPRT1 (Hs02800695_m1). mRNA levels were calculated using the SDS 2.1 software (Applied Biosystems).

Total RNA was extracted using Reliaprep™ extraction columns (Promega, Madison, USA), analyzed at Nanodrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA) in order to assess acid nucleic quantity and quality (using 260/280 nm and 260/230 nm ratios) and then retrotranscripted to obtain cDNA via a Superscript^® Vilo™ cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Transcripts were quantified using real-time quantitative PCR on an ABI PRISM 7900 sequence detector (Applied Biosystems, Foster City, CA, USA) with an Applied Biosystems predesigned TaqMan Gene Expression Assays and Absolute QPCR ROX mix (Thermo Fisher Scientific, Waltham, MA, USA). The following probes were used (Applied Biosystems, assay identification numbers in parentheses): TLR1 (Mm01208874_m1), TLR2 (Mm00442346_m1), TLR3 (Mm01207404_m1), TLR4 (Mm00445273_m1), TLR5 (Mm00546288_s1), TLR6 (Mm02529782_s1), TLR7 (Mm00446590_m1), TLR8 (Mm04209873_m1), and TLR9 (Mm00446193_m1). In each sample, mRNA quantity was normalized to the amounts of β2-microglobulin (Mm00437762_m1) mRNA. Relative mRNA quantities were expressed as fold over β2-microglobulin mRNA level.

Lymphocyte populations of interest were FACS sorted and collected. Total RNA was isolated by using TRIZOL (Life Technologies) according to the manufacturer’s instructions. RNA concentrations were determined using Nanodrop 1000 (Thermo Fisher Scientific). RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fischer) according to the manufacturer’s protocol (Applied Biosystems). qPCR was performed using SyberGreen Gene Expression Assay (Life Technologies) with the following primers: C-myc forward 5’-GCCCCCAAGGTAGTGATCCT-3’ and reverse 5’-GTGCTCGTCTGCTTGAATGG-3’ and Id3 forward 5’-GAAATCCTGCAGCGTGTCAT-3’ and reverse 5’-GTCAGTGGCAAAAGCTCCTC-3’. Gene expression was normalized as n-fold difference to the gene Hprt1 forward 5’-TGATCAGTCAACGGGGGACA-3’ and reverse 5’-TTCGAGAGGTCCTTTTCACCA-3’. qPCR reaction was performed on a ABI Prism 7900 sequence detector (Applied Biosystems, Foster City, CA).

RNA was converted into cDNA and then measured by quantitative real-time PCR using ABI PRISM 7900 Sequence Detector (Applied Biosystems, Foster City, CA, USA). The PCR product was amplified to obtain a statistically significant increase in target genes, which was determined using the threshold cycle (Ct) and normalized with GADPH [25 (link)]. The primer sequences are shown in Table 2.

A 4-mm coronal section was either taken from the ipsilateral or the contralateral cortices of injured or sham animals at days 1 and 4 post-injury. After extraction with the use of RNeasy Mini Kits (QIAGEN, Valencia, CA), RNA samples were subjected to reverse transcription with SuperScript II RNase H reverse transcriptase (Invitrogen, Carlsbad, CA). Realtime quantitative RT-PCR analysis was performed with an ABI PRISM 7900 sequence detector (Applied Biosystems, Foster City, CA). Thermal cycling was initiated with a 2-min incubation at 50°C, followed by a 10-min denaturation step at 95°C and 40 cycles at 95°C for 15 s and 60°C for 1 min. The primers and probes for BDNF (TaqMan Gene Expression Assay ID 00432069-ml) and β-actin (Rn00607939_s1) were obtained from Applied Biosystems. Relative quantities of the BDNF and β-actin mRNA were calculated with the previously described comparative threshold cycle method [25] (link).