Mir 99a 5p

Nine miRNA were chosen for RT-qPCR validation; miR-29a-3p (TaqMan® ID 007600_mat, Applied Biosystems), miR-99a-5p (TaqMan® ID 006254_mat), miR-126-3p (TaqMan® ID 008451_mat), miR-140-3p (TaqMan® ID 471823_mat), miR-222-3p (TaqMan® ID 000525), miR-223-3p (TaqMan® ID 002295), miR-204-5p (TaqMan® ID 000508), miR-409-3p (TaqMan® ID 002332), miR-6119-5p (Custom TaqMan® small RNA Assay). Reverse transcription was achieved on 10 ng of total RNA using the TaqMan® MicroRNA Reverse Transcription (Applied Biosystems, Foster City, CA, USA) kit following the manufacturer’s instructions. In the thermal cycler (StepOne+, Applied Biosystems, Foster City, CA, USA), each 15 μL RT reaction followed 30 min at 16°C, 30 min at 42°C, 5 min at 85°C. Then, 1.3 μL of miRNA-specific cDNA from the reaction were amplified using the TaqMan® Small RNA Assays (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Amplification was performed at 95°C for 10 min, pursued by 40 cycles of 95°C for 15 s and 60°C for 1 min. All miRNA levels were normalized to the values of U6 snoRNA [47 (link),48 (link)].

Seven miRNA were chosen for RT-qPCR validation among known miRNA which expression was affected by the food deprivation and based on their level of expression; miR-99a-5p (TaqMan^® ID 006254_mat, Applied Biosystems), miR-126-3p (TaqMan^® ID 008451_mat), miR-140-3p (TaqMan^® ID 471823_mat), miR-222-3p (TaqMan^® ID 000525), miR-223-3p (TaqMan^® ID 002295), miR-204-5p (TaqMan^® ID 000508), miR-409-3p (TaqMan^® ID 002332). Reverse transcription was achieved on 10 ng of total RNA using the TaqMan^® MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. In the thermal cycler (StepOne+, Applied Biosystems, Foster City, CA, USA), each 15 μL RT reaction followed 30 min at 16°C, 30 min at 42°C and 5 min at 85°C. Then, 1.3 μL of miRNA-specific cDNA from the reaction were amplified using TaqMan^® Small RNA Assays (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Amplification was performed at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. All miRNA levels were normalized to the values of U6 snoRNA [30 , 31 (link)] and the results expressed as fold changes of threshold cycle (Ct) values relative to the control using the 2^-ΔΔCt method [32 (link)].