Amv reverse transcriptase kit

Northern blotting protocol was adapted from Summer et al. (2009) (link) and http://archive.bio.ed.ac.uk/ribosys/protocols/website_Northern_blotting.pdf. 5 μg of Trizol extracted RNAs or 1 μg of immunoprecipitated RNAs was separated on 4% urea PAGE against 0.5×— TBE buffer at 25 W for 90 min. RNAs were transferred to Amersham Hybond-NX membrane (#RPN203T; GE Healthcare) for 2 hr at 65 V, UV-cross-linked, blocked for 1 hr in SES buffer (0.5 M Na₃PO₄ pH7.2, 7% [wt/vol] SDS, 1 mM EDTA), and hybridized O/N at 37°C with radiolabelled α-satellite probes (end-labeling method using primer extension-system AMV reverse transcriptase kit, #E3030; Promega, Madison, WI) diluted in SES buffer. Membrane was washed in 6×— SSPE (1.08 M NaCl, 0.06 M NaH₂PO₄, 20 mM EDTA, pH adjusted to 7.4) two times for 15 min at 37°C and two times for 30 min at 42°C. Blot was exposed to P32-sensitive film (Hyblot CL film, #E3012; Denville, Saint Laurent, Canada) at −80°C to reveal the potential interaction for a short (<24 hr) or long (>24 hr) period of time. The sequences of the radiolabeled probes are indicated in Supplementary file 10.

Total cellular RNA was extracted using Trizol reagent (Invitrogen Corporation, CA, USA), and cDNA was synthesized by using the AMV Reverse Transcriptase kit (Promega, Madison, WI). The mRNA quantification methods were as described [42 (link), 43 (link)]. The primer sequences were listed in (Supplementary Table 1).

Total RNA was extract by using the TripleXtractor reagent (Bio-Cell) as indicated by the supplier, and the AMV Reverse Transcriptase kit (Promega) was used to produce cDNA following the manufacturer’s recommendations, by using 2 μg of total RNA. Quantitative PCR reactions were performed by using the Bio-Rad CFX96 qPCR thermocycler. The primer pair sequences for selected amplicons were designed using the online IDT PrimerQuest Tool software (IDT; https://eu.idtdna.com/Primerquest/Home/Index). Primer sequences are available in Supplementary Table 1. Gapdh mRNA level was used as an internal control and results were expressed as previously described^{56 (link)}.

Trizol reagent (Invitrogen, Burlington, ON, Canada) was used to isolate total RNA, as indicated by the supplier. The AMV Reverse Transcriptase kit (Promega) was used to generate cDNA following the manufacturer’s recommendations. Quantitative PCR reactions were performed by using the CFX96 thermocycler (Bio-Rad Laboratories, CA, USA). Supplementary Table 1 shows the primers sequence for all amplicons, designed by using the online IDT PrimerQuest Tool software (IDT, Integrated DNA Technologies Inc., IA, USA; https://eu.idtdna.com/Primerquest/Home/Index). Results were normalized by using mouse GAPDH as internal control^{11 (link)}.

Total cellular RNA was extracted using Trizol reagent (Invitrogen Corporation, CA, USA), and cDNA was synthesized by using the AMV Reverse Transcriptase kit (Promega, Madison, WI). The mRNA quantification methods were as described.^{23 (link),24 (link)} The primer sequences were listed in Supplementary Table S2

Reverse transcription was performed from 1 μg of total RNA in a reaction volume of 20 μl, containing 0.4 mM Oligo(dT)₁₅ primer, 2 mM dNTP mix, 5 mM MgCl2, 20 U RNase inhibitor and 10 U AMV reverse transcriptase (AMV Reverse Transcriptase kit, Promega). The mixture was incubated 10 min at room temperature (RT), followed by incubations: one hour at 42°C, 5 min at 99°C and finally 10 min on ice. Then, the PCR was performed in a personal Robocycler Gradient 96 (Stratagene) in a reaction volume of 50 μl and comprised 2 μl of cDNA, 5 μl of 10X enzyme buffer, 10 μl of 5X GCmelt buffer (Ozyme), 200 nM of each primer, 0.4 mM of dNTP mix and 5 U Platinum Taq High fidelity DNA polymerase (Life Technologies). The program was applied as follows: 1 cycle of 5 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 54-57°C, 4 min at 68°C; 1 cycle of 10 min at 68°C. Following PCR, migration on agarose gel was performed to visualize the amplified fragment and check the size.