Trizol chloroform method

Cells were collected by centrifugation, and RNA was extracted using the Trizol/chloroform method (Invitrogen) according to manufacturer’s instructions. The concentration of total RNA was measured by a ND-1000 NanoDrop spectrophotometer and one microgram of RNA was treated with DNAse I (Invitrogen) and used for the reverse transcription using the high capacity cDNA reverse transcription kit (Applied Biosystems). The resulting cDNA was diluted 1:4 and assessed by qRT-PCR using Power Syber Green Master Mix (Applied Biosystems). The volume of each reaction was 25 μL. Measurements were done in a 7500 Real Time PCR system. For each sample, qRT-PCR reactions were done in triplicate, and the entire analysis was done twice independently. Ct-values for each sample and the data were exported to Microsoft Excel for further analysis. The average Ct-value for the endogenous control (GADPH) was calculated for each sample. To calculate the relative expression of the gene of interest the delta-delta Ct-method was used [45 (link)]. The sequence of the primers used is provided in the Additional file 1: Table S1.

The effect of sublethal oxidative stress on the activation of senescence-associated secretory phenotype (SASP) was tested by means of gene expression of interleukin-6 (IL-6), interleukin-8 (IL-8), matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 3 (MMP3), and matrix metalloproteinase 13 (MMP13). These genes are typically expressed in a proinflammatory and catabolic environment during inflammation-related disc degeneration [44 (link), 45 (link)]. IVD cells were seeded on 6-well plates and senescence was induced as described above (n = 4). After 24 hours, RNA was extracted with the Trizol/chloroform method according to the manufacturer's instructions (15596-018, Invitrogen, Carlsbad, CA, USA) and 1 μg was reverse transcribed to cDNA using a reverse transcription kit (4374966, Applied Biosystems). cDNA was then mixed with primers and master mix (4352042, Applied Biosystems) and gene expression was measured using real-time PCR. The following primers were used: TATA box binding protein (TBP) Hs00427620_m1, IL-6 Hs00174131_m1, IL-8 Hs00174103_m1, MMP1 Hs00233958_m1, MMP3 Hs00968308_m1, and MMP13 Hs00233992_m1. Data was analyzed with the comparative Cq method (2^−ΔΔCq, housekeeping gene TBP). Results are presented as a fold change relative to the untreated control group.

7–9 week old C57BL/6J male mice brains were freshly frozen in OCT compound and sectioned at 14 μM on Leica 3050 s cryostat, taking every other section throughout the hippocampus. Sections were then mounted on PEN membrane slides, ~12 sections per slide with 3 slides per brain. The staining and preparation of sections for the LCM procedure was done using the LCM staining kit (Cat# AM1935, Life Technologies) with Cresyl Violet following manufacturer's recommendations. Following staining, slides were kept at room temperature before beginning LCM. The microdissection of the CA1, CA3 and DG hippocampal regions were done using the Arcturus PixCell II LCM microscope where the laser power was set at 50 mW and each capture was done with 2 or fewer laser pulses. After dissection, each sample was placed in 50 μl ice-cold Trizol and RNA was extracted using the Trizol-Chloroform method (Life technologies). Purified RNA from each dissection was subjected to a single round of linear T7 RNA Polymerase–driven transcription using MessageAmp™ II aRNA Amplification Kit (Cat# AM1751, Life Technologies).