RNeasy Plus Mini Kit

Protocol tips

Upstream tips	Protocol tips	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.

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

HNSCC cells were seeded in 60 mm dishes (2 × 105 cells/dish) and supernatants were collected and saved for secreted protein analysis. Cells were washed twice with PBS before isolating total RNA using RNeasy Plus mini kit (QIAGEN) as per manufacturer's protocol. 500 ng of isolated RNA was then reverse transcribed to cDNA using iScript cDNA synthesis kit (Bio‐Rad) and a thermocycler under the following conditions: 5 min at 25° C, 30 min at 42° C, and 5 min at 85° C. The resultant cDNA samples were used to perform quantitative (i.e. real time) PCR analysis in a 96‐well optical plate with each well containing 6 μL of cDNA, 7.5 μL of SyBr Green Universal SuperMix (Bio‐Rad), and 1.5 μL oligonucleotide primers (forward and reverse; 4 μM) for a total reaction volume of 15 μL) on an ABI PRISM Sequence Detection System (model 7000, Applied Bio systems) with the following protocol: 95 °C for 15 s (denaturing) and 60 °C for 60 s (annealing) repeated for 40 cycles. Oligonucleotide primers were obtained from Integrated DNA Technologies (IDT; Coralville, IA) and described in Supplementary Table 1. Relative gene expression was determined by the comparative 2ˆ (−ΔΔCT) method. Briefly, cycle threshold (CT) values of all genes were normalized to that of GAPDH for each sample (in duplicate) and then fold changes were determined by comparing the normalized CT values of erlotinib‐sensitive with erlotinib‐resistant HNSCC cells. Each assay was performed in triplicate (i.e. on three separate occasions) and the results were presented as mean ± standard error of the mean.

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Manufacturer protocol

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