RevertAid RT Reverse Transcription Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The enzyme maintains activity at 42-50 °C and is suitable for synthesis of cDNA up to 13 kb.	Use following conditions: 25°C for 5 min, 55°C for 30 min and 75°C for 10 min	The reverse transcription reaction product can be directly used in PCR applications or stored at -20 °C for less than one week. For longer storage, -70 °C is recommended

Upstream tips
The enzyme maintains activity at 42-50 °C and is suitable for synthesis of cDNA up to 13 kb.
Protocol tips
Use following conditions: 25°C for 5 min, 55°C for 30 min and 75°C for 10 min
Downstream tips
The reverse transcription reaction product can be directly used in PCR applications or stored at -20 °C for less than one week. For longer storage, -70 °C is recommended

Publication protocol

Total RNA was extracted from plasma (1 ml) or MG-63 and U2OS cells (3×104 cells/ml) using RNAzol® RT RNA Isolation Reagent (GeneCopoeia, Guangzhou, China). RNA concentrations were measured using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNA extraction was repeated until all RNA samples had an A260/A280 ratio between 1.8 and 2.0. cDNA was synthesized using RevertAid RT Reverse Transcription kit (Thermo Fisher Scientific, Inc.) under the following conditions: 25°C for 5 min, 55°C for 30 min and 75°C for 10 min. One-Step PrimeScript RT-PCR kit for Real Time RT-PCR (Clontech Laboratories, Inc., Mountainview, CA, USA) was used to prepare all PCR reaction conditions. Primer sequences were as follows: LINK-A, forward 5′-TTCCCCCATTTTTCCTTTTC-3′, reverse 5′-CTCTGGTTGGGTGACTGGTT-3′; GAPDH, forward 5′-GAAGGTGAAGGTCGGAGT-3′, reverse 5′-GAAGATGGTGATGGGATTTC-3′. qPCR reaction conditions were: Initial denaturation at 95°C for 50 sec, followed by 40 cycles of 95°C for 15 sec and 60.5°C for 34 sec. Relative expression levels were quantified using the 2−ΔΔCq method (13) and normalized to GADPH loading control.

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Manufacturer protocol

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