Absolute Quantification of RNA Transcripts

Selfie RT-qPCR that measures the absolute number of RNA transcripts per gene was performed as described by Podlesniy and Trullas [33 (link)]. Briefly, cells were extracted and diluted in the 100ST buffer (DireCtQuant, Lleida, Spain, DCQ100ST) following the manufacturer’s instructions. Then, a pre-annealing step was performed per duplicate, by mixing 2 μl of the sample with the same volume of 2.5 μM of the corresponding reverse primer (Trem2 5′-ctcggagactctgacactgg-3′; Clec7a 5′-gcactgcagcaaccactact-3′) at 70 °C for 5 min. Next, the reaction mixture containing 1 mM dNTP mixture, 10 U of the RiboLock RNase Inhibitor (Thermo Fisher Sci., EO0381) and 200 U of Maxima H Minus Reverse Transcriptase (Thermo Fisher Sci., EP0751) or 50% glycerol was retro-transcribed for 30 min at 60 °C followed by 5 min at 85 °C. Finally, 2.5 μM of the corresponding forward primer (Trem2 5′-tggaaccgtcaccatcactc-3′; Clec7a 5′-cttcaccttggaggcccatt-3′) was added and amplified by conventional RT-qPCR (5 min at 95 °C, followed by 49 cycles of 15 s at 95 °C, 25 s at 60 °C and 25 s at 72 °C), using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 1725271). The number of transcripts per encoding gene was calculated as the fold change after subtracting the Ct values obtained from the sample containing glycerol.