Agilent DNF-472 HS RNA Quick Start Guide

Analytical specifications

HS RNA assay (15nt)

Sizing Range

200 nt – 6,000 nt

Sizing Accuracy1

+ 20%

Sizing Precision1

20% CV

Limit of Detection (S/N > 3)

50 pg/µL – Total RNA 250 pg/µL –mRNA

Quantitative Range (per smear)

50 pg/µL – 5,000 ng/µL – Total RNA 500 pg/µL – 5,000 pg/µL mRNA

Quantification Accuracy1

+ 30%

Quantification Precision1

20% CV

Physical Specifications3

Total electrophoresis run time

22cm: 31 minutes, 33cm: 40 minutes, 55cm: 70 minutes

Samples per run

12, 48 or 96; depending on the instrument type

Sample volume required

2 µL

Kit stability

4 months

Agilent DNF-472 HS RNA (15 nt) Kit Quick Guide for Fragment Analyzer Systems

The Agilent Fragment Analyzer systems are automated capillary electrophoresis platforms for scalable, flexible, fast, and reliable electrophoresis of nucleic acids.

This Quick Guide is intended for use with the Agilent 5200, 5300, and 5400 Fragment Analyzer systems only. This kit is designed to analyze Total RNA at a concentration of 50 pg/μL – 5,000 pg/μL input sample concentration and

mRNA at an input sample concentration of 250 pg/μL – 5,000 pg/μL .

Specifications

1,2

Results using RNA Ladder as sample and 33-55 capillary array.

Results using Total RNA and ribo-depleted mRNA samples diluted in nuclease-free water.

DNF-472 HS RNA Kit (15 nt) Quick Guide for Fragment Analyzer Systems

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Kit Components – 500 Sample Kit

Kit Component Number

Part Number (Re-order Number)

Description Quantity Per Kit

5191-6574*

DNF-265-0240 RNA Separation Gel, 240 mL 1

DNF-301-0008 BF-1 Blank Solution, 8mL 1

DNF-355-0125 5x 930 dsDNA Inlet Buffer, 125 mL

DNF-497-0125 0.25x TE Rinse Buffer, 125 mL 1

DNF-472-FR*

DNF-600-U030

DNF-370-0004

DNF-386-U015 HS RNA Ladder, 15 μL 1

5191-6612* Quantitative DNA, RT

C275-130 Eppendorf LoBind 0.5 mL tubes (bag of 50) 1

DNF-475-0050 5x Capillary Conditioning Soln, RT 1

HS RNA (15 nt), 500, 4oC

• Dilute with sub-micron filtered water prior to use

HS RNA (15 nt) FR

Intercalating Dye, 30 μL

HS RNA Diluent Marker (15 nt), 4 mL

• Dilute with sub-micron filtered water prior

to use

*Not orderable.

WARNING

Refer to product safety data sheets for further information

When working with the Fragment Analyzer kit components follow the appropriate safety procedures such as wearing goggles, safety gloves and protective clothing.

DNF-472 HS RNA Kit (15 nt) Quick Guide for Fragment Analyzer Systems

to use

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Kit Components – 1000 Sample Kit

Kit Component Number

Part Number (Re-order Number)

Description Quantity Per Kit

5191-6575*

DNF-265-0500 RNA Separation Gel, 500 mL 1

DNF-301-0008 BF-1 Blank Solution, 8mL 1

DNF-355-0300 5x 930 dsDNA Inlet Buffer, 300 mL

DNF-497-0250 0.25x TE Rinse Buffer, 250 mL 1

DNF-472-FR*

DNF-600-U030

DNF-370-0004

DNF-386-U015 HS RNA Ladder, 15 μL 2

5191-6613* HS RNA (15 nt), 1000, RT

C275-130 Eppendorf LoBind 0.5 mL tubes (bag of 50) 1

DNF-475-0100 5x Capillary Conditioning Soln, RT 1

HS RNA (15 nt), 1000, 4oC

• Dilute with sub-micron filtered water prior

HS RNA (15 nt), FR

Intercalating Dye, 30 μL

HS RNA Diluent Marker (15 nt), 4 mL

• Dilute with sub-micron filtered water prior

to use

*Not orderable.

WARNING

Refer to product safety data sheets for further information

When working with the Fragment Analyzer kit components follow the appropriate safety procedures such as wearing goggles, safety gloves and protective clothing.

NOTE: RNA samples and RNA Ladders are very sensitive to RNase contamination, which can lead to experimental failure. To

minimize RNase contamination, wear gloves when working with RNA samples and reagents, and when handling accessories that will come in contact with the RNA sample. Use certified RNase-free plastics and disposable consumables. It is also recommended to work in a separate lab space if possible and decontaminate the pipettes and work surface to avoid cross contamination.

DNF-472 HS RNA Kit (15 nt) Quick Guide for Fragment Analyzer Systems

Additional Material Required for Analysis with the Fragment Analyzer Systems

• Fragment Analyzer systems with LED fluorescence detection:

• 5200 Fragment Analyzer system (p/n M5310AA)

• FA 12-Capillary Array Ultrashort, 22 cm (p/n A2300-1250-2247) OR

• FA 12-Capillary Array Short, 33 cm (p/n A2300-1250-3355) OR

• FA 12-Capillary Array Long, 55 cm (p/n A2300-1250-5580)

• 5300 Fragment Analyzer system (p/n M5311AA)

• FA 48-Capillary Array Short, 33 cm (p/n A2300-4850-3355) OR

• FA/ZAG 96-Capillary Array Short, 33 cm (p/n A2300-9650-3355) OR

• FA/ZAG 96-Capillary Array Long, 55 cm (p/n A2300-9650-5580)

• 5400 Fragment Analyzer system (p/n M5312AA)

• FA 48-Capillary Array Short, 33 cm (p/n A2300-4850-3355) OR

• FA/ZAG 96-Capillary Array Short, 33 cm (p/n A2300-9650-3355) OR

• FA/ZAG 96-Capillary Array Long, 55 cm (p/n A2300-9650-5580):

• Agilent Fragment Analyzer controller software (Version 1.1.0.11 or higher)

• Agilent ProSize data analysis software (Version 2.0.0.61 or higher)

Additional equipment/reagents required (not supplied)

• 96-well PCR sample plates. Please refer to Appendix – Fragment Analyzer Compatible Plates and Tubes in the Fragment Analyzer System User Manual for a complete approved sample plate list

• Multichannel pipettor(s) and/or liquid handling device capable of dispensing 1 – 100 µL volumes (sample plates) and 1,000 µL volumes (inlet buffer plate)

• Pipette tips

• 96-well plate centrifuge (for spinning down bubbles from sample plates)

• Sub-micron filtered DI water system (for diluting the 5x 930 dsDNA Inlet Buffer and 5x Capillary Conditioning Solution)

• 96-deepwell 1mL plate: Fisher Scientific #12-566-120 (inlet buffer and/or waste plate)

• Reagent reservoir, 50 mL (VWR #89094-680 or similar) (for use in pipetting inlet buffer plates/sample trays)

• Conical centrifuge tubes for prepared separation gel/dye mixture and/or 1x Capillary Conditioning Solution

• 50 mL (for 5200 Fragment Analyzer system or 50 mL volumes): BD Falcon #352070, available from Fisher

Scientific #14-432-22 or VWR #21008-940

• 250 mL (for 5300 and 5400 Fragment Analyzer systems or larger volumes): Corning #430776, available from Fisher Scientific #05-538-53 or VWR #21008-771

• Vortexer (for mixing of samples, ladders, and/or markers in tubes and/or plates)

Capillary Storage Solution (p/n GP-440-0100)

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