M mlv reverse transcriptase buffer

For qPCR experiments, total RNA was extracted from cells using the RNeasy® Mini kit (Qiagen; 74106) according to the manufacturer's instructions. RNA quality was checked by spectral analysis (A260/ 280 nm), and then samples were stored at −80°C. Reverse transcription was performed using the M-MLV reverse transcriptase (Invitrogen; 28025013; 5U/μL final concentration), 500ng total RNA, a random hexamer primer (Thermo Scientific, GER; S0142; 10ng/μL final concentration), and DNTPs (Roche, CH; 1 277 049; 5mM final concentration) in M-MLV reverse transcriptase buffer (Invitrogen; 18057-018), for a total volume of 20μL. SYBR Green-based quantitative PCR was performed using the LightCycle® 480 SYBR Green I master reaction mix (Roche, 04707516001), 10 ng of cDNA, and the LightCycler 480 real-time PCR system (Roche) (40 cycles of amplification). Raw data (Ct values) were analyzed using the comparative Ct method. Gene expression data were calculated as relative to the expression of housekeeping genes. The comparative threshold cycle method (ΔΔCT) was used to quantify relative gene expression, and the obtained quantification was transformed to exponential 2−ΔΔCT values. A p-value below 0.05 (Student’s t-test) was considered as statistically significant. Primers sequences used for RT-qPCR are listed in Supplementary Table 1.

Total RNA was extracted from the uteri and marrow derived dendritic cells (BMDCs) with Trizol^® reagent (Invitrogen, Carlsbad, CA) and was reverse transcribed into stable cDNA using M‐MLV reverse transcriptase buffer (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions. The amplification of cDNA was performed using 2× UltraSYBR Mixture (CWBIO, Co. Ltd, Beijing, China). Real‐time quantitative PCR was carried out with a LightCycler 480 PCR instrument (Roche, Indianapolis, IN). The target gene mRNA expression was normalized to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) expression. The fold change was calculated as 2^−ΔΔCt (cycle threshold). The primers used for quantitative PCR are listed in Table S1.

cDNA was generated from 1 μg RNA, using 1 μg random primers (48190011, Invitrogen), 50U M-MLV reverse transcriptase (Invitrogen), 1 mM dNTPs (Invitrogen) in M-MLV reverse transcriptase buffer (Invitrogen) supplemented with 5 mM DTT (Thermofisher). The cDNA mix was heated for 45′ at 42 °C and then for 3′ at 99 °C.
Q-RT-PCR was performed as described in Horos et al.^{4 (link)}, with the following modifications. A master mix was created using 10 μM of each primer, 12.5 μl SYBR Green master mix (4309155, Applied Biosystems) and 5 μl cDNA filled to a final concentration of 20 μl. Primers can be found in Supplementary Table S-I.