RNA HS Assay Kit——StarLighter

Description

Accurate measurement of low-concentration RNA can be difficult when traditional UV A260 readings are affected by limited sensitivity or by substances commonly present in laboratory samples. The StarLighter RNA HS Assay Kit provides a high-sensitivity fluorescent RNA quantification method for measuring initial RNA concentrations from 0.5 ng/µL to 100 ng/µL, with a total assay range of 5–100 ng.

This RNA HS Assay Kit is highly selective for RNA over double-stranded DNA and tolerates specified concentrations of common contaminants, including salts, proteins, free nucleotides, solvents and detergents. It provides an accurate solution for laboratories that need to quantify low-abundance RNA using a Qubit fluorometer or fluorescence microplate reader.

The assay accepts 1–20 µL of sample in a final volume of 200 µL. After mixing, tubes or microplates are incubated for 2 minutes at room temperature while protected from light. The fluorescent signal remains stable for up to 3 hours after incubation when protected from light.

Two configurations are available: FS-T1006-S for 100 reactions and FS-T1006 for 1000 reactions. The kit contains RNA HS Buffer QB, RNA HS Standard QS0, RNA HS Standard QS1 and RNA HS Fluorescent Reagent QF.

Specification

Kit Components

Materials Required but Not Supplied

• Qubit Fluorometer or fluorescence microplate reader
• Thin-walled, clear 0.5 mL PCR tubes for Qubit Fluorometer measurements
• Black 96-well microplate for fluorescence microplate reader measurements
• Vortex mixer
• Pipettes and sterile, RNase-free pipette tips

Features

• High-sensitivity RNA quantification: Measures initial RNA concentrations from 0.5 ng/µL to 100 ng/µL and total RNA amounts from 5–100 ng.
• RNA-selective fluorescence detection: Highly selective for RNA over double-stranded DNA.
• Flexible sample input: Accepts 1–20 µL of RNA sample in a final assay volume of 200 µL.
• Rapid assay workflow: Requires a 2-minute incubation at 20–25°C while protected from light.
• Stable fluorescent signal: Signal remains stable for up to 3 hours after incubation when protected from light.
• Multiple instrument options: Compatible with a Qubit fluorometer or fluorescence microplate reader.
• 96-well assay capability: Can be performed in a black-wall, clear-bottom 96-well microplate using 620 nm excitation and 660 nm emission.
• Contaminant tolerance: Tolerates specified concentrations of proteins, salts, solvents, detergents, agarose, PEG and dNTPs.
• Two available sizes: Supplied in 100-reaction and 1000-reaction configurations.

Tested Contaminant Tolerance

For the tested contaminants, “OK” usually represents less than 10% perturbation under the concentrations specified above.

Application

• High-sensitivity fluorescent RNA quantification
• Measurement of RNA samples with initial concentrations from 0.5–100 ng/µL
• Quantification of total RNA amounts from 5–100 ng
• RNA concentration measurement using a Qubit fluorometer
• RNA quantification using a fluorescence microplate reader
• 96-well fluorescence-based RNA analysis
• Research laboratory RNA measurement

Qubit Fluorometer Assay Setup

1. Equilibrate all kit components to room temperature.
2. Prepare the working solution by diluting RNA HS Fluorescent Reagent QF 1:200 in RNA HS Buffer QB. For example, combine 11 µL QF with 2189 µL QB.
3. Do not prepare or mix the working solution in a glass container.
4. Add 190 µL of working solution and 10 µL of the appropriate standard to each standard tube.
5. Add 180–199 µL of working solution and 1–20 µL of sample to each sample tube.
6. Keep the final volume of every assay tube at 200 µL.
7. Vortex vigorously for 3–5 seconds.
8. Incubate for 2 minutes at room temperature while protected from light.
9. Read the two standards and samples using the RNA High Sensitivity assay setting.

Fluorescence Microplate Reader Assay Setup

1. Equilibrate all components to room temperature and mix thoroughly by vortexing.
2. Prepare the working solution by combining RNA HS Fluorescent Reagent QF and RNA HS Buffer QB at a 1:199 ratio.
3. Prepare a standard curve from 0–10 ng/µL by serially diluting RNA HS Standard QS1.
4. Add 10 µL of each standard or 1–20 µL of each sample to its designated well.
5. Adjust the working solution volume so that every well contains exactly 200 µL.
6. Mix the plate for 10–15 seconds.
7. Incubate for 2 minutes at room temperature while protected from light.
8. Measure fluorescence at 620 nm excitation and 660 nm emission.
9. Calculate unknown RNA concentrations from the standard curve. Dilute and retest samples that exceed the linear range.

FAQ