Agilent DNF-471 RNA Quick Start Guide

Analytical specifications

RNA kit (15nt)

Sizing Range

200 nt – 6,000 nt

Sizing Accuracy1

+ 5%

Sizing Precision1

5% CV

Limit of Detection (S/N > 3)

5 ng/µL

Qualitative Range (per smear)

5 ng/µL – 500 ng/µL

Quantitative Range (per smear)

25 ng/µL – 500 ng/µL

Quantification Accuracy1

+ 20%

Quantification Precision1

10% CV

Physical Specifications3

Total electrophoresis run time

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-471 RNA Kit (15 nt) 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 detect Total RNA within the range of 5 ng/μL to 500 ng/μL input sample concentration and IVT RNA in the range of 1 ng/ μL to 100ng/ μL

Specifications

1,2,3

Results using RNA Ladder as sample.

DNF-471 RNA (15 nt) Quick Guide for Fragment Analyzer Systems

WARNING

•

Kit Components – 500 Sample Kit

Kit Component Number

Part Number (Re-order Number)

Description Quantity Per Kit

5191-6572*

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

DNF-300-0008 BF-25 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-471-FR*

DNF-600-U030

DNF-369-0004

DNF-382-U020 RNA Ladder, 20 μL 5

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

*Not orderable.

RNA (15 nt), 500, 4oC

• Dilute with sub-micron filtered water prior to use

RNA (15 nt), FR

Intercalating Dye, 30 μL

RNA Diluent Marker (15 nt), 4 mL

• Dilute with sub-micron filtered water prior

to use

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-471 RNA (15 nt) Quick Guide for Fragment Analyzer Systems

•

Kit Components – 1000 Sample Kit

Kit Component Number

Part Number (Re-order Number)

Description Quantity Per Kit

5191-6573*

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

DNF-300-0008 BF-25 Blank Solution, 8mL 1

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

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

DNF-471-FR*

DNF-600-U030

DNF-369-0004

DNF-382-U020 RNA Ladder, 20 μL 10

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

*Not orderable.

RNA (15nt), 1000, 4oC

• Dilute with sub-micron filtered water prior to use

RNA (15 nt), FR

Intercalating Dye, 30 μL

RNA Diluent Marker (15 nt), 4 mL

• Dilute with sub-micron filtered water prior

to use

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-471 RNA (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)

•

DNF-471 RNA (15 nt) Quick Guide for Fragment Analyzer Systems

Keep reagents during sample preparation at room temperature

Ensure that no sample or Diluent Marker remains within or on the outside of the tip

Essential Measurement Practices

Environmental conditions

Steps before sample preparation

Pipetting practice

• Ambient operating temperature: 19 – 25 °C (66 – 77 °F)

•

• Allow reagents to equilibrate at room temperature for 30 min prior to use

• Pipette reagents carefully against the side of the 96-well sample plate or sample

tube

•

RNA Ladder Preparation

Upon arrival of the ladder, it is recommended to divide the ladder into aliquots with working volume typical for one day use or one sample plate. Store aliquots in Eppendorf 0.5 mL LoBind tubes at -70°C or below.

1. Thaw RNA Ladder aliquot in PCR tube on ice.

2. Mix by pipetting the solution up and down with a pipette tip and spin down. Transfer the ladder to a RNase-free PCR tube. Heat-denature the ladder at 70°C for 2 min, immediately cool to 4°C and keep on ice.

RNA Sample Preparation

1. Heat-denature all total RNA samples at 70°C for 2 min if needed and immediately cool to 4°C and keep on ice before use.

2. The total RNA input sample must be within a total concentration range of 5 ng/µL to 500 ng/µL or 1 ng/µL to 100 ng/µL for IVT mRNA for optimal assay results. If the concentration of the sample is above this range, dilute with RNase-free water.

Sample Plate Preparation

1. Using a fresh RNase-free 96-well sample plate, pipette 22 μL of the RNA Diluent Marker (15 nt) (DM) Solution to each well in a row that is to contain sample or RNA Ladder. Fill any unused wells within the row of the sample plate with 24 μL/well of BF-25 Blank Solution.

2. Pipette 2 μL of each denatured RNA sample into the respective wells of the sample; mix the contents of the well using the pipette by aspiration/expulsion in the pipette tip.

3. RNA Ladder: The RNA Ladder must be run in parallel with the samples for each experiment to ensure the accurate

quantification. Pipette 2 μL of denatured RNA Ladder into the 22 μL of Diluent Marker (15 nt) (DM) Solution in Well 12 of each

row to be analyzed (12-capillary system) or Well H12 (96-capillary system). Mix the contents of the well using the pipette by aspiration/expulsion in the pipette tip.

4. After mixing sample/RNA Ladder and Diluent Marker (15 nt) Solution in each well, centrifuge the plate to remove any air bubbles. Check the wells of the sample plate to ensure there are no air bubbles trapped in the bottom of the wells. The presence of trapped air bubbles can lead to injection failures.

5. For best results, run the plate as soon as possible. If the sample plate will not be used immediately, cover the sample plate with RNase-free cover film, store at 4°C and use within 24 hours. Remove the cover film before placing the plate into the instrument.

6. To run the samples, place the plate in one of the three sample plate trays (Drawers 4-6 from the top) of the Fragment Analyzer instrument. Load or create the experimental method.

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