For the LSI setup, we collected by FACS sorting no more than 200 PKHpos and 200 PKHneg cells in 96-well plates in 10 µl of Single Cell Lysis Kit plus DNase (Ambion). Total RNAs were reverse-transcribed using Human Megaplex RT Primers mix, followed by preamplification with Human Megaplex PreAmp Primers (pool A). Then HT qPCR profiling was performed on TaqMan human platform A V2.1 (Applied Biosystems). We improved the original protocol from Applied Biosystems (Table 1).

Table 1.
Applied Biosystems’ HT qPCR profiling protocol as improved by the authors
Step or dilutionLSI (µl)10 ng Applied (µl)
RNA to RT 3 µl  10 ng 
RT (two reactions) 7.5 7.5 
Pooled RT reaction 15 15 
RT to PreAmp (preamplification) 5 5 
Final PreAmp 25 25 
PreAmp cycles 16 14 
Post-PreAmp dilution 1:4 1:4 
Dilution PCR 1:20 1:50 
Final dilution factor 1:80 1:200 
Step or dilutionLSI (µl)10 ng Applied (µl)
RNA to RT 3 µl  10 ng 
RT (two reactions) 7.5 7.5 
Pooled RT reaction 15 15 
RT to PreAmp (preamplification) 5 5 
Final PreAmp 25 25 
PreAmp cycles 16 14 
Post-PreAmp dilution 1:4 1:4 
Dilution PCR 1:20 1:50 
Final dilution factor 1:80 1:200 
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Raw data (i.e., cycle threshold [Ct] values) were exported to Excel (Microsoft). miRNAs with raw Ct >28 or not expressed (e.g., not amplified) were excluded from the analysis. Expressed miRNAs (Ct <28) were then normalized over the median of housekeeping controls (RNU44, RNU48, and RNU6B) for human array and over the median of U6b, SnoRNA135, and SnoRNA202 for rodent array. Regulated miRNAs were selected based on the following criteria: P value < 0.05, |log2 fold| > 0.5.

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