SuperScript IV VILO Master Mix

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Use up to 2.5 μg of total RNA as starting material in a 20-μL reaction	Treat the mixture at 25 °C for 10 min, incubation at 50 °C for 10 min, and incubation at 85 °C for 5 min.

Upstream tips
Use up to 2.5 μg of total RNA as starting material in a 20-μL reaction
Protocol tips
Treat the mixture at 25 °C for 10 min, incubation at 50 °C for 10 min, and incubation at 85 °C for 5 min.

Publication protocol

RAW 264.7 cells (500000 cells/well) were seeded in 6-well plates and treated for 4 h with LPS (1 µg/mL), with or without pre-incubation with EnP(5,8) for 2 h. Afterwards, the supernatant was removed and the cells were disrupted in 1 mL of PureZOL™ reagent. Then, the samples were transferred to a RNase-free tube and 0.2 mL of chloroform were added. The mixture was shaken vigorously for 15 s. After 5 min incubation at room temperature, the samples were centrifuged at 12,000× g for 15 min at 4 °C. Following centrifugation, the aqueous phase containing the RNA was immediately transferred to a new RNase-free tube and 0.5 mL of isopropyl alcohol were added, and mixture was incubated at room temperature for 5 min. Afterwards, the tubes were centrifuged at 12,000× g for 10 min at 4 °C, the RNA appearing as a white pellet on the side and bottom of the tube. Supernatant was carefully discarded and the RNA pellet was washed with 1 mL of 75% of ethanol. After vortexing, the mixture was centrifuged at 7500× g for 5 min at 4 °C and the supernatant was carefully discarded. Then, the RNA pellet was air-dried for about 5 min and reconstituted in 50 µL of PCR grade water. Subsequently, the RNA was quantified in a Qubit 4 fluorometer (Invitrogen by Thermo Fisher Scientific; Waltham, MA, USA), using the Qubit™ RNA HS assay kit. The RNA quality and integrity was then evaluated using the QubitTM RNA IQ assay kit. In order to obtain the complementary DNA, 1 µg of RNA was mixed with 4 µL SuperScript™ IV VILO™ Master Mix in a 20-µL reaction. The reverse-transcribed reaction involved three steps: incubation at 25 °C for 10 min, incubation at 50 °C for 10 min, and incubation at 85 °C for 5 min.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing cDNA synthesis Cell lines using SuperScript IV VILO Master Mix from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for SuperScript IV VILO Master Mix below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing cDNA synthesis Cell lines using SuperScript IV VILO Master Mix from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.