The V617F mutation was generated in the murine JAK2 cDNA using PCR mutagenesis, verified by DNA sequencing, and the JAK2 WT or V617F mutant cDNAs cloned into the MIG R1 vector [20] in the position 5' to the internal ribosome entry site.
A miR-148a overexpression vector was generated by amplifying a primary form of miR-148a from murine spleen cDNA using the following primers: pri148a forward 5′-GTTAACTGTGACATTGCCACCAGA-3′ and pri148a reverse 5′-CTCGAGAAAAAAACGACGTGGCCAACA-3′; and cloned under the control of U6 promoter into pQCXIX (BD Biosciences Clontech) using HpaI and XhoI restriction enzymes.
Total RNA samples were prepared from the desired leaf materials using RNeasy plant mini kit (Qiagen, Germany), and were converted into cDNAs using Moloney Murine leukemia virus (M-MLV) reverse transcriptase (Promega, USA).
Total RNA was extracted from lung and liver tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse-transcribed into cDNA using murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA, USA).
Total RNA was extracted from gut and lung tissues using a Trizol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse-transcribed into cDNA using murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA, USA).
One microgram of total RNA from mock and SY5Y-APP cells was transcribed into cDNA using murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) and oligo-dT (15 18) as a primer (final volume, 50 μl).
RNA concentrations were determined spectrophotometrically and then reverse transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen).
Subsequently, 0.5 μg of DNA-free RNA from each sample was used for the synthesis of first strand cDNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase according to the manufacturer's instructions (Invitrogen).
In brief, RNA was reversely transcribed to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen, Carlsbad, CA).
Total RNA (5 μg) was reverse transcribed to generate first-strand cDNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen) and random primer mix (Invitrogen).
