Agilent DNF-470 Small RNA Analyzer Quick Guide PDF

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Summary of Content for Agilent DNF-470 Small RNA Analyzer Quick Guide PDF

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Agilent DNF-470 Small RNA Kit Quick Guide for the 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. The Small RNA Kit (275 samples) (Part # DNF-470-0275) is designed for the quantification of Small RNA samples, and determination of microRNA region content. Synthetic RNA can be analyzed within the defined sizing region with this kit.

Specifications

Analytical specifications1,2 Small RNA assay

Sizing Range 15 nt 200 nt

Qualitative Range 25 pg/L 2500 pg/L (microRNA region)

Quantitative Range 50 pg/L 2000 pg/L

Quantification Precision 25% CV (Small RNA Ladder)

Physical Specifications3

Total electrophoresis run time 22cm2: 18 minutes, 33cm: 24 minutes, 55cm: 45 minutes

Samples per run 12, 48 or 96; depending on the instrument type1

Sample volume required 2 L

Kit stability 4 months

Recommended Sample Concentrations

MicroRNA 50 pg/L 2000 pg/L

Small RNA 1 ng/L 20 ng/L

Total RNA 5 ng/L 100 ng/L

1 Results using Total RNA, small RNA, and MicroRNA samples and fragments diluted in nuclease-free water. 2 The 22 cm effective, 47 cm total length capillary array is only available for 12-capillary Fragment Analyzer instruments.

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Kit Components 275 Sample Kit

Kit Component Number

Part Number

(Re-order Number)

Description Quantity Per Kit

5191-6571* Small RNA, 275, 4oC

DNF-262-0250 Small RNA Separation Gel, 250 mL 1

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

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

Dilute with sub-micron filtered water prior to use

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

DNF-470-FR* Small RNA, FR

DNF-600-U030 Intercalating Dye, 30 L 1

DNF-368-0004 Small RNA Diluent Marker, 4 mL 2

DNF-361-U060 Small RNA Ladder, 600 L 1

5191-6612* Quantitative DNA, RT

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

DNF-475-0050 5x Capillary Conditioning Soln, 50mL 1

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.

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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 L volumes (sample plates) and

1,000 L 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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Essential Measurement Practices

Environmental conditions

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

Keep reagents during sample preparation at room temperature

Steps before sample preparation

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

Pipetting practice

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

tube

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

Small RNA Diluent Marker Preparation

Remove the Small RNA Diluent Marker from -20oC and keep it on ice before use. Vortex the tube briefly to mix the content. Spin the tube after mixing to ensure liquid is at the bottom of the tube.

Small RNA Ladder Preparation

Prior to first use, the Small RNA Ladder solution should be aliquoted to minimize the number of freeze/thaw cycles.

Using the provided Eppendorf LoBind 0.5 mL tubes, aliquot 12 L of Small RNA Ladder per tube into 5 tubes and store

the aliquots at less than -70C. Each aliquot is good for 5 freeze/thaw cycles.

Thaw a Small RNA Ladder aliquot on ice.

Transfer a volume of the Small RNA Ladder for one day of use to an RNase-free PCR tube. Heat-denature the Small

RNA Ladder at 70C for 10 min using a thermal cycler, immediately cool to 4C and keep on ice before use.

RNA Sample Preparation

It is recommended to heat-denature RNA samples at 70C for 10 min and immediately cool to 4C and keep on ice before use, as needed.

If the concentration of the input sample is above the recommended range, dilute the sample with RNase-free water.

Sample Plate Preparation

Using a fresh RNase-free 96-well sample plate, pipette 18 L of the Small RNA Diluent Marker (DM) Solution to each well in a row that is to contain sample or Small RNA Ladder. Fill any unused wells within the row of the sample plate with 20 L/well of BF-25 Blank Solution.

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

Small RNA Ladder: The Small RNA Ladder must be run in parallel with the samples for each experiment to ensure for accurate quantification. Pipette 2 L of denatured Small RNA Ladder into the 18 L of Diluent Marker (DM) Solution in Well 12 of each row to be analyzed. Mix the contents of the well using the pipette by aspiration/expulsion in the pipette tip.

After mixing sample or Small RNA Ladder with Small RNA Diluent Marker 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.

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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 2-8C and use within the same day. Spin the plate again if any bubbles developed in the sample wells. Be sure to remove the cover film before placing the plate into the instrument.

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.

Important Sample Mixing Information:

When mixing sample with diluent marker, it is important to mix the contents of the well thoroughly to achieve the most accurate quantification. It is highly suggested to perform one of the following methods to ensure complete mixing:

When adding 2 L of sample or ladder to 18 L of diluent marker, swirl the pipette tip while pipetting up/down to further mix. OR

After adding 2 L of sample or ladder to 18 L of diluent marker, place a plate seal on the sample plate and vortex the sample plate at 3,000 rpm for 2 min. Any suitable benchtop plate vortexer can be used. Ensure that there is no well-to-well transfer of samples when vortexing. The plate should be spun via a centrifuge after vortexing to ensure there are no trapped air bubbles in the wells. OR

When adding 2 L of sample or ladder to 18 L of diluent marker, use a separate pipette tip set to a larger 18 L volume, and pipette each well up/down to further mix. OR

Use an electronic pipettor capable of mixing a 10 L volume in the tip after dispensing the 2 L sample volume. Some models enable using the pipette tip for both adding and mixing.

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Gel preparation

Prepare gel/dye mixture for 5200, 5300, and 5400 Fragment Analyzer Systems. To ensure the gel/dye mixture is mixed homogeneously without generating bubbles, gently invert the centrifuge tube 5 to 10 times, depending on the volume of the mixture. NOTE: Centrifuge dye prior to opening the vial to reduce risk of leaking.

5200 Fragment Analyzer system volume specifications

# of Samples to be Analyzed1

Volume of Intercalating Dye

Volume of Separation Gel2

Volume of 1x Conditioning Solution2

12 1.0 L 10 mL 10 mL

24 1.5 L 15 mL 15 mL

36 2.0 L 20 mL 20 mL

48 2.5 L 25 mL 25 mL

96 4.5 L 45 mL 45 mL

1 One sample well per separation is dedicated to the ladder. 2 A 5 mL minimum volume in the tube is included.

5300 Fragment Analyzer system volume specifications with 48-capillary array

# of Samples to be

Analyzed1

Volume of Intercalating

Dye

Volume of Separation

Gel2

Volume of 1x Conditioning

Solution2

48 2.5 L 25 mL 25 mL

96 4.0 L 40 mL 40 mL

144 5.5 L 55 mL 55 mL

192 7.0 L 70 mL 70 mL

240 8.5 L 85 mL 85 mL

288 10.0 L 100 mL 100 mL

1 One sample well per separation is dedicated to the ladder. 2 A 5 mL minimum volume in the tube is included.

5300 and 5400 Fragment Analyzer systems volume specifications with 96-capillary arrays

# of Samples to be Analyzed1

Volume of Intercalating Dye

Volume of Separation Gel2

Volume of 1x Conditioning Solution2

96 4.0 L 40 mL 40 mL

192 8.0 L 80 mL 80 mL

288 12.0 L 120 mL 120 mL

384 16.0 L 160 mL 160 mL

480 20.0 L 200 mL 200 mL

1 One sample well per separation is dedicated to the ladder.

2 A 5 mL minimum volume in the tube is included.

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Agilent Small RNA DNF-470 assay operating procedure

1.Mix fresh gel and dye according to the volumes in the Gel preparation tables. Refill 1x Capillary Conditioning Solution as needed.

2.Place a fresh 1x 930 dsDNA Inlet Buffer in drawer B on the system, 1.0 mL/well. Replace daily.

2.1. 5200 system; Fill row A of buffer plate

2.2. 5300 system - 48 capillary; Fill rows A-D of buffer plate

2.3. 5300/5400 system - 96 capillary; Fill all rows of buffer plate

3.Prepare Capillary Storage Solution plate. Replace every 2-4 weeks for optimal results.

3.1. 5200 system; Fill row H of buffer plate with 1.0mL/well, place in drawer B

3.2. 5300 system - 48 capillary; Fill rows A-D of a sample plate with 100 L/well, place in drawer 3

3.3. 5300/5400 system - 96 capillary; Fill all rows of a sample plate with 100 L/well, place in drawer 3

3.3.1. 5400 system; place in drawer S

4.Place 0.25x TE Rinse Buffer plate in drawer M on the system, 200 L/well. Replace daily.

4.1. 5200 system; Fill row A of sample plate

4.2. 5300 system - 48 capillary; Fill rows A-D of sample plate

4.3. 5300/5400 system - 96 capillary; Fill all rows of sample plate

5.Mix samples or Ladder with Diluent Marker in sample plate, add 20 L of BF-25 Blank Solution to unused wells. Place ladder in corresponding well dependent on the capillary size.

WARNING Working with Chemicals

The handling of reagents and chemicals might hold health risks.

Refer to product material safety datasheets for further chemical and biological safety information.

Follow the appropriate safety procedures such as wearing goggles, safety gloves and protective clothing.

5200 system; Ladder well 12, depending on which row is chosen

5300 system - 48 capillary; Ladder well D12 or H12, depending on which group is chosen

5300/5400 system - 96 capillary; Ladder well H12

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Agilent Fragment Analyzer software operating procedure

1.Select Row, Group or Tray to run.

2.Enter sample ID and Tray ID (optional).

3.Select Add to Queue, from the dropdown menus select the corresponding method based on your capillary length;

3.1 DNF-470-22 Small RNA

3.2 DNF-470-33 Small RNA

3.3 DNF-470-55 Small RNA

4.Enter Tray Name, Folder Prefix, and Notes (optional).

5.Select OK to add method to the queue.

6.Select to start the separation.

Small RNA Ladder result

Representative Small RNA Ladder result using Fragment Analyzer system with the DNF-470 Small RNA Analysis kit. Method: DNF-470-33. Peaks annotated by size (nt).

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Troubleshooting

The following table lists several potential assay specific issues which may be encountered on occasion when using the Small RNA Analysis kit and suggested remedies. Contact Agilent technical support if you have any additional troubleshooting or maintenance questions.

Issue Cause Corrective Action

Sample and/or ladder signal too weak or degraded.

1 Sample and/or ladder degraded. 2 Sample, ladder and/or diluent

marker are contaminated. 3 Sample concentration is too low and

out of range. 4 Sample not added to Diluent Marker

solution or not mixed well 5 Rinse buffer is not fresh or a wrong

rinse buffer used. 6 Array was contaminated.

1 Use fresh sample and/or ladder. 2 Clean working area and equipment

with RNaseZap. Always wear gloves when preparing sample/ladder. Use new sample, ladder aliquot and diluent marker.

3 Verify sample was within

concentration range specified for the Small RNA Analysis kit and prepare sample at high concentration; OR Repeat experiments using increased injection time and/or injection voltage.

4 Verify sample was correctly added

and mixed to sample well. 5 Prepare a new rinse buffer plate with

200 L/well 0.25x TE Rinse Buffer. 6 Follow the Method C outlined in

Capillary Array Cleaning of the Fragment Analyzer User Manual to decontaminate and clean the capillary array.

Sample signal drops abruptly at the end of separation.

1 Sample concentration too high and out of range.

1 Verify sample was within concentration range specified for the Small RNA Analysis kit.

Missing LM signal or noisy baselilne.

1 Expired Diluent Marker solution.

2 Dirty array inlet.

3 Aging array.

1 Use a fresh Diluent marker solution.

2 Follow Method C outlined in

Capillary Array Cleaning of the

Fragment Analyzer User Manual to

clean the array.

3 Replace the array with a new array.

If issue persists, contact Agilent

Technical Support.

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Manualsnet FAQs

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