RNeasy Mini Kit

RNA isolation / purification Tissue - Human Mouth

Experiment
RNA isolation / purification Tissue - Human Mouth
Product
RNeasy Mini Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

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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Discussion

Discussion

1 year ago

Author: Paul G. Macon United States

RNA isolation from tissue

How do I extract RNA from animal tissue without using liquid nitrogen? I tried the RNA extraction by using the TRIzol reagent and I homogenize the tissue using polytron homogenizer at room temperature for 30secs is this correct?

Discussion

2 years ago

Author: Aaron Stege Netherlands

Problem in phase separation after using serum/plasma kit

I used a serum/plasma kit for my serum samples. After the phase separation the samples should have 3 phases: a colourless aqueous phase, a white interphase and a red organic phase. However, in some of my samples there was no aqueous phase unless I wait for an extended period of time. How can I circumvent this problem?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Tissue - Human Mouth using RNeasy Mini Kit from Qiagen.

Paper title
Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics
Full paper
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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy Mini Kit below.

Download PDF Download manufacturer protocol

Videos

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