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. - For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

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. - For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

Publication protocol

Total RNA was isolated from CHO or UPS-1 cells using Isogen (Nippon Gene) or RNeasy Mini Kit (Qiagen) and then subjected to RT-PCR analysis using the SuperScript III One-Step RT-PCR system (Invitrogen). For quantitative RT-PCR (qRT-PCR), total RNA was subjected to reverse transcription using a SuperScript VILO cDNA Synthesis Kit (Invitrogen). The resultant cDNA was used as a template for PCR using LightCycler FastStart DNA MasterPLUS SYBR Green I (Roche Applied Science); fold changes in gene expression were normalized to the β-actin mRNA level. Chinese hamster ATP11C cDNA was amplified using the following primer pairs: set 1 (sense, 5′-ATACTGAGCTCTTAGAACTGACC-3′; antisense, 5′-ATCA­CTATTCCTGGCTGCTTGG-3′), set 2 (sense, 5′-GAACAGCACA­TCA­ACGTTGATAC-3′; antisense, 5′-CTGATAAATATGAGGAGAATTATGG-3′), and set 3 (3′ untranslated region (UTR); sense, 5′-GTATAGGGTTCAGAATAAATGTCC-3′; antisense, 5′-GATATTAGACCAAGACAATTAGTC-3′). The ATP11A, CDC50A, and β-actin cDNAs were amplified using the following primer pairs: sense, 5′-CATGGAAGTGCTCAA­GAGAGAC-3′/antisense, 5′-GAGCAGGCTGACAGTGACAA­G-3′; sense, 5′-GCCA­GTTAAATGGAGACCCTAG-3′/antisense, 5′-GTCC­AGCTGGTAATGTTGGATG-3′; and sense, 5′-CTGTATGCCTCTGGTCGTAC-3′/antisense, 5′-GCCATCTCCTGCTCGA­AGTC-3′, respectively.

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Manufacturer protocol

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