RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Protocol tips
- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Publication protocol

Total RNA was isolated from gingival tissue biopsies of ten subjects with and without periodontitis (six patients with periodontitis and four healthy controls) as previously described17 using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The amount of total RNA was quantified using a Qubit spectrophotometer (Molecular Probes, Eugene, Oregon, USA). cDNA synthesis was performed from 1 µg of total RNA per 20 µl of reaction using the iScript™ cDNA Synthesis Kit (BioRad, Herkules, CA, USA), according to the manufacturer’s instructions. The gene expression analysis was performed by quantitative reverse transcription PCR (qRT-PCR) using TaqMan Gene Expression Assays together with TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). TaqMan probes were used as follows: IGLL5 (Hs04330879_u1), SSR4 (Hs00196721_m1), MZB1 (Hs00414907_m1), XBP1 (Hs00231936_m1) and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs02758991_g1). All qRT-PCR reactions were run in duplicates on the 7500 Fast Real-Time PCR system (Applied Biosystems). Gene expression was calculated according to the ΔΔCt method, where each periodontitis sample was normalized to the healthy control sample and to GAPDH (reference gene) of corresponding sample. Statistical significance was calculated using Wilcoxon signed rank test and p < 0.05 was considered significant.

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Manufacturer protocol

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