Rnase free glycogen

HIO RNA isolation was performed by applying 500 µL TRIzol™ reagent (Invitrogen, Washington, DC, USA; 15596026) to each well and disrupting the monolayer mechanically by pipetting. Monolayers were incubated for 5 min at RT and transferred to Eppendorf tubes. An amount of 100 µL chloroform was added and the tubes were shaken for 15 s and placed at RT for 3 min. The cell lysates were centrifuged at 12,000× g for 15 min at 4 °C and the upper aqueous phases were transferred to new RNase-free 1.5 mL Eppendorf tubes. An amount of 1 µL of 20 mg/mL Rnase-free glycogen (Invitrogen) and 250 µL isopropanol (Sigma) were added, and tubes were mixed vigorously for 15 s. The homogenous solutions were incubated for 10 min at RT and were centrifuged at the previously described settings. Supernatants were discarded and RNA pellets were washed in 500 µL 75% ethanol 3 times with tube inversion, and subsequently centrifuged for 5 min, 7500× g at 4 °C. After removing the supernatant, RNA pellets were air dried for 5–10 min and eluted in 20 µL Rnase-free water. RNA was quantified using the NanoDrop One (Thermo Scientific). Pellets were purified with Dnase according to the manufacturer’s recommendations (Qiagen, Hilden, Germany, 1023460), eluted in 15 µL Rnase-free water, and stored at −80 °C.

Total RNA of pollinated and parthenocarpic litchi fruits was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol^{78 (link)}. Initially, 100 mg of litchi fruit was homogenized in 1 mL of TRIzol reagent (Invitrogen) and was centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was transferred to a new clean tube, incubated at room temperature for 5 min, mixed with 0.2 mL of chloroform and shaken vigorously for 15 s. After an incubation for 3 min at room temperature, the sample was centrifuged at 12,000 × g for 15 min at 4 °C and the aqueous phase was transferred to a new clean tube. Then, 10 µg of RNase-free glycogen (Invitrogen) was added to the tube, followed by the addition of 0.5 mL of 100% isopropanol and incubation for 10 min. After the sample was centrifuged at 12,000 × g for 10 min at 4 °C, the resulting RNA sample was washed and stored at −80 °C.

Rnase R and RNasin Inhibitor were supplied by Epicenter (Madison, WI). SuperScript™ III Reverse Transcriptase, TRIzol, 5×RT Buffer solution, RNase-Free water, and RNase-Free glycogen were obtained from Invitrogen Life Technologies (Carlsbad, CA). dNTP Mix was supplied by HyTest Ltd. (Turku, Finland). Ultrapure water was supplied by a Milli-Q water purification system (Bedford, MA). All other chemicals and solvents used were of at least analytical grade.

Total RNA from small EV CD81⁺ was extracted with Trizol (Invitrogen), according to the manufacturer’s instructions. Before precipitating the RNA with isopropyl alcohol, 20 μg RNase-free glycogen (Invitrogen) was added to the aqueous phase and the samples were stored for 16h at −80°C. RNA pellets were dissolved in RNase-free water and quantified by Qubit (Invitrogen). About 25 ng of total RNA were retrotranscribed and preamplified. Amplified cDNA products were diluted in 75 μL of RNase-free purified water and loaded onto microfluidic cards of the TaqMan Human MicroRNA Array Av2.0 (Applied Biosystems). Quantitative RT-PCR reactions were performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems) as follows: 94.5 °C for 10 min, followed by 40 amplification cycles of 97°C for 30 sec and 59.7°C for 1 min.

THP-1 cells were cultured in RPMI1640 (Invitrogen) supplemented with 10% FBS, penicillin/streptomycin (Invitrogen), 10 mM HEPES (Invitrogen), 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol. HeLa cells were cultured in Eagle’s MEM (Invitrogen) supplemented with 10% FBS, 1% NEAA (Invitrogen), and penicillin/streptomycin. Total-cell lysates were harvested in TRIzol reagent (Invitrogen); total RNA was purified from TRIzol lysates according to the manufacturer’s instructions; and we used RNase-free glycogen (Invitrogen) as a carrier in the aqueous phase prior to precipitating the RNA with isopropyl alcohol. The RNA extracts were checked by an Agilent 2100 Bioanalyzer, which confirmed their qualities as RIN scores 9.6 and 9.9 for the THP-1 and HeLa RNA extracts. The prepared total RNAs were mixed with the ratio described above. The same RNA extracts previously described (Kanamori-Katayama et al. 2011 (link)) were used.

TRI reagent BD (MRCGENE), isopropanol, and 100% ethanol were obtained from Shanghai Chemical Reagent Co., Ltd., and glacial acetic acid from Sinopharm Group Chemical Reagent Co., Ltd. RNase-free glycogen, SuperScript” III Reverse Transcriptase, and 5× RT buffer were obtained from Invitrogen Life Technologies, and RNase inhibitor from Epicenter Inc. dNTP mixture (dATP, dGTP, dCTP, and dTTP, 2.5 mM) were obtained from HyTest Ltd. Primer (HyTest Ltd.).
A Clean Bench (Shanghai Bo Xun Industrial Co. Ltd. Medical Equipment Factory), DK-8D electric heating constant-temperature water tank (Shanghai Senxin Experimental Instrument Co., Ltd.), and a Gene Amp PCR System 9700 (Applied Biosystems) were used forLncRNA gene chip detection was performed using an Arraystar human lncRNA chip V3.0 chip.