Microplate Assays for Nucleic Acids

Accurate, efficient nucleic acid quantitation is critical for cellular and molecular biology labs that work with precious, rare, or difficult-to-process samples and applications requiring precise measurement. These applications include next-generation sequencing, transfection, real-time PCR, measuring yields of RNA preparations for microarray experiments, reverse transcription PCR (RT-PCR), differential display PCR, northern blot analysis, S1 nuclease assays, RNase protection assays, and cDNA library preparations.

dsDNA assays

PicoGreen reagent assays provide a rapid and simple fluorescence-based method for the accurate quantitation of dsDNA. The assays are many times more sensitive than conventional UV absorbance and can be optimized for different concentration ranges. PicoGreen reagent assays are routinely used for genotyping by allele-specific PCR, quantitating dsDNA samples before and after PCR amplification, and determining PCR amplification yields before sequencing.

ssDNA assays

OliGreen reagent assays provide a rapid and simple fluorescence-based method for the accurate quantitation of ssDNA. The assay is many times more sensitive than conventional UV absorbance, and free nucleotides or short oligonucleotides of up to 6 bases do not interfere with the OliGreen ssDNA quantitation assay. OliGreen reagent assays are routinely used for antisense oligonucleotides, aptamers, genomic DNA isolated under denaturing conditions, PCR primers, sequencing primers, and single-stranded phage DNA.

RNA assays

RiboGreen reagent assays provide a rapid and simple fluorescence-based method for the accurate quantitation of RNA. RiboGreen reagent does not detect significant sample contamination by free nucleotides and thus more accurately measures the amount of intact RNA in potentially degraded samples. The RiboGreen reagent is ideal for fast and accurate measurement of RNA before generating cDNA, northern blot analysis, S1 nuclease assays, RNase protection assays, reverse transcription PCR, and differential display PCR.

Linear quantitation of calf thymus DNA from 25 pg/mL to 1,000 ng/mL using PicoGreen dsDNA Quantitation Reagent. Samples were excited at 480 nm, and the fluorescence emission intensity was measured at 520 nm and plotted as a function of DNA concentration. The inset shows the results obtained with DNA concentrations between 0 and 750 pg/mL.

Selection guides

	Quant-IT dsDNA Assay Kit, high sensitivity	Quant-iT dsDNA Assay Kit, broad range	Quant-iT 1X dsDNA high sensitivity
Target	dsDNA	dsDNA	dsDNA
Quantitation range	0.2–100 ng	2-1,000 ng	0.1–100 ng
Ex / Em (nm)	502 / 523	510 / 527	502 / 523
Components	dsDNA HS fluorescent reagent, dilution buffer and standards	dsDNA BR fluorescent reagent, dilution buffer and standards	1x working solution and standards
Format	1,000 assays	1,000 assays	1,000 assays
Protocol outline	Prepare working solution by diluting fluorescent reagent into buffer Add working solution to wells Add unknown DNA sample Measure fluorescence Quantitate the dsDNA by comparing to standard curve		Add 1X working solution to wells Add unknown DNA sample Measure fluorescence Quantitate the dsDNA by comparing to standard curve
Cat. No.	Q33120	Q33130	Q33232

	Quant-iT OliGreen ssDNA Reagent	Quant-iT OliGreen ssDNA Assay Kit
Target	ssDNA	ssDNA
Ex / Em (nm)	500 / 525	500 / 525
Sample	Selectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution	Selectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution
Quantification range	Detect 200 pg to 2 µg ssDNA in a 200 µL assay volume	Detect 200 pg to 2 µg ssDNA in a 200 µL assay volume
Components	Bulk reagent	Includes buffer and DNA standard
Assay format	2,000 assays using a 200 µL assay volume	2,000 assays using a 200 µL assay volume
Protocol outline	Dilute samples in microplate wells. Add working solution of the OliGreen reagent. Incubate at room temperature for 2–5 min. Measure fluorescence. Quantitate the ssDNA by comparing to standard curve.
Cat. No.	O7582	O11492

	Quant-iT RiboGreen RNA Assay Kit	Quant-iT RNA Assay Kit	Quant-iT RNA Assay Kit, broad range	Quant-iT microRNA Assay Kit	Quant-iT RNA HS Reagent
Target	RNA	RNA	RNA	Small RNA	RNA
Quantitation range	1–200 ng	5–100 ng	20–1,000 ng	1–100 ng	5–100 ng
Ex / Em (nm)	500 / 525	644 / 673	644 / 673	500 / 525	644 / 673
Components	Fluorescent RiboGreen RNA reagent, RNase-free buffer and rRNA standards	Fluorescent RNA reagent, RNase-free buffer and rRNA standards	Fluorescent RNA BR reagent, RNase-free BR buffer and rRNA standards	Fluorescent microRNA reagent, RNase-free microRNA buffer and microRNA standards	Fluorescent RNA HS reagent
Protocol Outline	Dilute samples in microplate wells Add RNA reagent working solution Incubate to room temperature for 2–5 minutes Measure fluorescence Quantitate the RNA by comparing to standard curve
Cat. No.	R11490	Q33140	Q10213	Q32882	Q32884

dsDNA assays

	Quant-IT dsDNA Assay Kit, high sensitivity	Quant-iT dsDNA Assay Kit, broad range	Quant-iT 1X dsDNA high sensitivity
Target	dsDNA	dsDNA	dsDNA
Quantitation range	0.2–100 ng	2-1,000 ng	0.1–100 ng
Ex / Em (nm)	502 / 523	510 / 527	502 / 523
Components	dsDNA HS fluorescent reagent, dilution buffer and standards	dsDNA BR fluorescent reagent, dilution buffer and standards	1x working solution and standards
Format	1,000 assays	1,000 assays	1,000 assays
Protocol outline	Prepare working solution by diluting fluorescent reagent into buffer Add working solution to wells Add unknown DNA sample Measure fluorescence Quantitate the dsDNA by comparing to standard curve		Add 1X working solution to wells Add unknown DNA sample Measure fluorescence Quantitate the dsDNA by comparing to standard curve
Cat. No.	Q33120	Q33130	Q33232

ssDNA assays

	Quant-iT OliGreen ssDNA Reagent	Quant-iT OliGreen ssDNA Assay Kit
Target	ssDNA	ssDNA
Ex / Em (nm)	500 / 525	500 / 525
Sample	Selectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution	Selectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution
Quantification range	Detect 200 pg to 2 µg ssDNA in a 200 µL assay volume	Detect 200 pg to 2 µg ssDNA in a 200 µL assay volume
Components	Bulk reagent	Includes buffer and DNA standard
Assay format	2,000 assays using a 200 µL assay volume	2,000 assays using a 200 µL assay volume
Protocol outline	Dilute samples in microplate wells. Add working solution of the OliGreen reagent. Incubate at room temperature for 2–5 min. Measure fluorescence. Quantitate the ssDNA by comparing to standard curve.
Cat. No.	O7582	O11492

RNA assays

	Quant-iT RiboGreen RNA Assay Kit	Quant-iT RNA Assay Kit	Quant-iT RNA Assay Kit, broad range	Quant-iT microRNA Assay Kit	Quant-iT RNA HS Reagent
Target	RNA	RNA	RNA	Small RNA	RNA
Quantitation range	1–200 ng	5–100 ng	20–1,000 ng	1–100 ng	5–100 ng
Ex / Em (nm)	500 / 525	644 / 673	644 / 673	500 / 525	644 / 673
Components	Fluorescent RiboGreen RNA reagent, RNase-free buffer and rRNA standards	Fluorescent RNA reagent, RNase-free buffer and rRNA standards	Fluorescent RNA BR reagent, RNase-free BR buffer and rRNA standards	Fluorescent microRNA reagent, RNase-free microRNA buffer and microRNA standards	Fluorescent RNA HS reagent
Protocol Outline	Dilute samples in microplate wells Add RNA reagent working solution Incubate to room temperature for 2–5 minutes Measure fluorescence Quantitate the RNA by comparing to standard curve
Cat. No.	R11490	Q33140	Q10213	Q32882	Q32884

Expertly detect fluorescence with Thermo Scientific plate readers

High-sensitivity fluorescence detection for 96-1,536 samples can be quickly performed on Fluoroskan or Fluoroskan FL Microplate Fluorometer or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents for optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.

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