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.

   
PicoGreen dsDNA Quantitation Reagent
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 sensitivityQuant-iT dsDNA Assay Kit, broad rangeQuant-iT 1X dsDNA high sensitivity
TargetdsDNAdsDNAdsDNA
Quantitation range0.2–100 ng2-1,000 ng0.1–100 ng
Ex / Em (nm)502 / 523510 / 527502 / 523
ComponentsdsDNA HS fluorescent reagent, dilution buffer and standardsdsDNA BR fluorescent reagent, dilution buffer and standards1x working solution and standards
Format1,000 assays1,000 assays1,000 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.Q33120Q33130Q33232
 Quant-iT OliGreen ssDNA ReagentQuant-iT OliGreen ssDNA Assay Kit
TargetssDNAssDNA
Ex / Em (nm)500 / 525500 / 525
SampleSelectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solutionSelectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution
Quantification rangeDetect 200 pg to 2 µg ssDNA in a 200 µL assay volumeDetect 200 pg to 2 µg ssDNA in a 200 µL assay volume
ComponentsBulk reagentIncludes buffer and DNA standard
Assay format2,000 assays using a 200 µL assay volume2,000 assays using a 200 µL assay volume
Protocol outline
  1. Dilute samples in microplate wells.
  2. Add working solution of the OliGreen reagent.
  3. Incubate at room temperature for 2–5 min.
  4. Measure fluorescence.
  5. Quantitate the ssDNA by comparing to standard curve.
Cat. No.O7582O11492
 Quant-iT RiboGreen RNA Assay KitQuant-iT RNA Assay KitQuant-iT RNA Assay Kit, broad rangeQuant-iT microRNA Assay KitQuant-iT RNA HS Reagent
TargetRNARNARNASmall RNARNA
Quantitation range1–200 ng5–100 ng20–1,000 ng1–100 ng5–100 ng
Ex / Em (nm)500 / 525644 / 673644 / 673500 / 525644 / 673
ComponentsFluorescent RiboGreen RNA reagent, RNase-free buffer and rRNA standardsFluorescent RNA reagent, RNase-free buffer and rRNA standardsFluorescent RNA BR reagent, RNase-free BR buffer and rRNA standardsFluorescent microRNA reagent, RNase-free microRNA buffer and microRNA standardsFluorescent RNA HS reagent
Protocol Outline
  1. Dilute samples in microplate wells
  2. Add RNA reagent working solution
  3. Incubate to room temperature for 2–5 minutes
  4. Measure fluorescence
  5. Quantitate the RNA by comparing to standard curve
Cat. No.R11490Q33140Q10213Q32882Q32884
dsDNA assays
 Quant-IT dsDNA Assay Kit, high sensitivityQuant-iT dsDNA Assay Kit, broad rangeQuant-iT 1X dsDNA high sensitivity
TargetdsDNAdsDNAdsDNA
Quantitation range0.2–100 ng2-1,000 ng0.1–100 ng
Ex / Em (nm)502 / 523510 / 527502 / 523
ComponentsdsDNA HS fluorescent reagent, dilution buffer and standardsdsDNA BR fluorescent reagent, dilution buffer and standards1x working solution and standards
Format1,000 assays1,000 assays1,000 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.Q33120Q33130Q33232
ssDNA assays
 Quant-iT OliGreen ssDNA ReagentQuant-iT OliGreen ssDNA Assay Kit
TargetssDNAssDNA
Ex / Em (nm)500 / 525500 / 525
SampleSelectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solutionSelectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution
Quantification rangeDetect 200 pg to 2 µg ssDNA in a 200 µL assay volumeDetect 200 pg to 2 µg ssDNA in a 200 µL assay volume
ComponentsBulk reagentIncludes buffer and DNA standard
Assay format2,000 assays using a 200 µL assay volume2,000 assays using a 200 µL assay volume
Protocol outline
  1. Dilute samples in microplate wells.
  2. Add working solution of the OliGreen reagent.
  3. Incubate at room temperature for 2–5 min.
  4. Measure fluorescence.
  5. Quantitate the ssDNA by comparing to standard curve.
Cat. No.O7582O11492
RNA assays
 Quant-iT RiboGreen RNA Assay KitQuant-iT RNA Assay KitQuant-iT RNA Assay Kit, broad rangeQuant-iT microRNA Assay KitQuant-iT RNA HS Reagent
TargetRNARNARNASmall RNARNA
Quantitation range1–200 ng5–100 ng20–1,000 ng1–100 ng5–100 ng
Ex / Em (nm)500 / 525644 / 673644 / 673500 / 525644 / 673
ComponentsFluorescent RiboGreen RNA reagent, RNase-free buffer and rRNA standardsFluorescent RNA reagent, RNase-free buffer and rRNA standardsFluorescent RNA BR reagent, RNase-free BR buffer and rRNA standardsFluorescent microRNA reagent, RNase-free microRNA buffer and microRNA standardsFluorescent RNA HS reagent
Protocol Outline
  1. Dilute samples in microplate wells
  2. Add RNA reagent working solution
  3. Incubate to room temperature for 2–5 minutes
  4. Measure fluorescence
  5. Quantitate the RNA by comparing to standard curve
Cat. No.R11490Q33140Q10213Q32882Q32884

Expertly detect fluorescence with Thermo Scientific plate readers

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High-sensitivity fluorescence detection for 6-1,536 samples can be quickly performed on the Varioskan ALF or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents to enable 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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