RNeasy Fibrous Tissue Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- This kit purifies RNA from up to 30 mg tissue		- If there is low RNA yield,Repeat RNA elution, incubate the RNeasy Mini spin column on the benchtop for 10 min with RNase-free water before centrifuging. - If there is low A260/A280 value use 10 mM Tris·Cl, pH 7.5, not RNase-free water, to dilute the sample before measuring purity. - If RNA does not perform well in downstream experiments this could be due to salt carryover dyuring elution step, so ensure that Buffer RPE is at 15–25°C.

Upstream tips
- This kit purifies RNA from up to 30 mg tissue
Downstream tips
- If there is low RNA yield,Repeat RNA elution, incubate the RNeasy Mini spin column on the benchtop for 10 min with RNase-free water before centrifuging. - If there is low A260/A280 value use 10 mM Tris·Cl, pH 7.5, not RNase-free water, to dilute the sample before measuring purity. - If RNA does not perform well in downstream experiments this could be due to salt carryover dyuring elution step, so ensure that Buffer RPE is at 15–25°C.

Publication protocol

Frozen uterine tissue ≤30 mg from CD-1 control and 30 ppm BPA exposure groups were homogenized for RNA isolation using an RNeasy Fibrous Tissue Mini Kit (Qiagen; Valencia, CA). Uterine RNA was converted to cDNA using a High Capacity cDNA Reverse Transcription Kit (Life Technologies; Grand Island, NY). Predesigned TaqMan RT-PCR assays (Applied Biosystems; Grand Island, NY) were used for the following genes: Col1a1 (Mm00801632_gH), Col3a1 (Mm00802296_g1), Mmp2 (Mm00439498_m1), Timp1 (Mm00441818_m1), Timp2(Mm00441825_m1), and Timp3(Mm00441826_m1). PCR amplification was performed in a final volume of 20 μL containing 1x TaqMan expression assay primers, Universal Master Mix (Applied Biosystems; Grand Island, NY), and 10 ng of cDNA. Amplification was performed in triplicates on a Step One Plus Real-Time PCR System (Applied Biosystems; Grand Island, NY) under the following fast conditions: 20 seconds at 95°C for one holding stage, then 1 second at 95°C and 20 seconds at 60°C for the cycling stage (60 cycles). Relative expression was quantified using the ΔΔCt method, using 18s as the endogenous control for normalization.

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Manufacturer protocol

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