Paris kit protein and rna isolation system

RNA from the nucleus or cytoplasm of cell lines was isolated by the PARIS™ Kit Protein and RNA Isolation System (Invitrogen). The qPCR method was used to detect the expression level of MAGI2-AS3 in the cytoplasm and nucleus. Nuclear control and cytoplasmic control used were U6 and GAPDH, respectively.

Nucleus and cytoplasm RNA were individually extracted from chicken myoblast during proliferation or differentiation phase according to the guide of PARIS Kit Protein and RNA Isolation System (Invitrogen, Carlsbad, CA, USA). The PARIS method is based on differential lysis of plasma and nuclear cell membranes by nonionic detergents. Cells are first separated into nuclear and cytoplasmic fractions then RNA is isolated. Briefly, the cell pellet was suspended in a buffer of RNA purification and then centrifuged twice at 4°C for 5 min. The supernatant was the cytoplasmic fraction and the pellet was the nucleus fraction. RNAs were extracted from both fractions using Trizol (Mange, Guangzhou, China). The effectiveness of cell fractionation can be easily checked by Quantitative PCR (qRT-PCR) or western blot with antibodies for proteins found predominantly in the nucleus or cytoplasm of cells such as sno-U6 (predominantly in the nucleus) or GAPDH (predominantly in the cytoplasm). In this study, we analyzed the sno-U6 and GAPDH expression with qRT-PCR to indicate the methodology was successful. The relative levels of GHR-AS and GHR-S were analyzed by qRT-PCR.

The nuclear and cytoplasmic fractions of Eca109 and Kyse150 cells were isolated according to the protocol of the PARIS™ Kit Protein and RNA Isolation System (Invitrogen). Total RNA was isolated simultaneously from independent cytoplasmic and nuclear fractions. Finally, qRT‐PCR was used to determine the relative expression of SNHG17 to estimate the relative ratios of SNHG17 in nuclear and cytoplasmic RNA concentrations.