Services
– Agilent Bioanalyzer
DNA-High Sensitivity Kit
This kit is kept in stock at the Genomics Core; other types of kits (see Other Bioanalyzer Kits) can be purchased from Agilent. Visit the Agilent website for product specifications of its DNA and RNA kits; highlights for the DNA HS kit are described below.
– Separate tubes with hinges are preferred.
– Tubes with narrow bottoms are preferred.
– Do
- Preferred Option: Provide exactly 1-µl of each sample (or replicate sample) per tube, as discussed in Loading Agilent Bioanalyzer chips... Genomics Core style!. Note: With the Preferred Option, duplicates are simply a second tube containing another µl of the same sample.
- Discouraged Option: This option is allowed, but definitely discouraged, as it involves more effort and chances for error. For each sample, provide 2-3 extra microliters to allow for accurate pipetting of a single µl. For running singlets, that’s 3-4 µl total; for duplicates, it would be 5-6 µl (and so on).
For a well-defined DNA smear (e.g., ~50-75 bp wide), a decent reading often can be achieved at concentrations as low as ~100 pM. For broad smears, 5 ng/µl is likely the absolute maximum rational concentration and values of ~1-3 ng/µl will likely be more suitable; see the following two images for examples with broad smears.
Bioanalyzer_Concentration_Guidelines_1.jpg; Bioanalyzer_Concentration_Guidelines_2.jpg
If you don’t already have a good idea of the DNA sample concentrations, it can be beneficial to determine if dilutions are needed by estimating sample concentrations with the Genomics Core's Qubit dsDNA kit (or, even a Nanodrop... as long as you pay attention to the caveats noted at Nanodrop Nucleic Acid Guide.pdf). You might also consider running serial dilutions on the chip to ensure that at least one well is within the right range.
Small RNA kit
The Small RNA kit is recommended for analysis of small/micro RNA molecules (e.g., separation of miRNA smears from tRNA). As we do not stock the Small RNA kit, you will need to coordinate with the Core at least 2-3 weeks ahead of when your small RNA samples would be ready for analysis.
RNA 6000 Pico kit
This kit is used for mRNA, Plant RNA, and Eukaryote or Prokaryote Total RNA.
– If you expect to run a large number of chips (e.g., ~5 chips... or ~55 samples) at one time, please notify the Core at least two weeks ahead to allow for ordering a new kit should stock be low.
– Preparing to run an RNA Bioanalyzer chip takes >1-2 hours; thus, samples are more likely to be run the same day if submitted early in the day (i.e., before noon – by 1:00 p.m., at the latest).
– If next-day service is okay, please state that in your Client Memo of the online submission; further, state whether you would prefer that the samples be refrigerated or frozen until run.
– Separate tubes with hinges are preferred.
– Tubes with narrow bottoms are preferred.
– Do
- Preferred Option: Provide exactly 1-µl of each sample (or replicate sample) per tube, as discussed in Loading Agilent Bioanalyzer chips... Genomics Core style!. Note: With the Preferred Option, duplicates are simply a second tube containing another µl of the same sample.
- Discouraged Option: This option is allowed, but definitely discouraged, as it involves more effort and chances for error. For each sample, provide 2-3 extra microliters to allow for accurate pipetting of a single µl. For running singlets, that’s 3-4 µl total; for duplicates, it would be 5-6 µl (and so on).
There are two reasons to skip sample heat-denaturation:
– "Hidden Breaks": Many species (especially among protostomes – e.g., arthropods, molluscs, annelids) contain a "Hidden Break" in the large subunit molecule (23S, 25S, or 28S RNA). Heating the purified RNA (or even some extraction methods) may cause the large subunit to separate into two roughly equal-size fragments that co-migrate during electrophoresis with the small subunit molecule (16S or 18S RNA). If so, that will result in an erroneously low RIN value. For species that contain a "hidden break", omitting the heat denaturation step may result in a more normal RNA profile.
The list below provides some examples of non-protostome organisms known to have "hidden breaks"; clients are responsible for determining if their particular organism might have "hidden breaks":
– RNase contamination: If your samples contain RNase molecules (either from lack of purification or through contamination), the heat-denaturation step may cause further RNA degradation, resulting in a poor Bioanalyzer profile. By contrast, keeping the samples cold until loaded in the chip may give a better view of the RNA integrity following your extraction protocol. However, please note that such residual RNase activity will ultimately lead to poor downstream results... but, you will not be aware of the problem because you skipped the heat-denaturation step. So, there is a trade-off when omitting the heat-denaturation step.
Online Submission Request
- Make the necessary 'selections'.
- Quantity: refers to the 'chip' (not your 'samples').
- In "Client Memo", specify:
(1) source of samples (e.g., prokaryotes, plants, animals);
(2) number of samples (& names, if desired); and,
(3) if same-day service is critical or if next-business-day service is okay (if so, also specify whether samples should be refrigerated or frozen if not run the same day).
For RNA samples (see RNA 6000 Pico kit), also specify:
(a) 'm RNA' or 'total RNA'; and,
(b) whether or not samples should be heat-denatured prior to analysis (see 'Sample Denaturation' comments above). - Click the 'check box', and submit your request.
Physical Submission of Samples
- Submission #;
- Date;
- PI's Last Name;
- Submitter name or initials; and,
- Special processing requests (if any).
Data Analysis
Other Bioanalyzer Kits
Click links to see examples of data (for a single sample) from some of the many assays that are available with the Agilent Bioanalyzer.- DNA_High-Sensitivity.jpg
- DNA_1000.jpg
- DNA_7500.jpg
- DNA_12000.jpg
- mRNA (nano-series).jpg
- mRNA (pico-series).jpg
- Small RNA (series II).jpg
- Plant RNA (nano).jpg
- Prokaryote Total RNA (pico).jpg
- Eukaryote Total RNA (nano).jpg
- Protein (High Sensitivity).jpg
- Protein (80 series).jpg
- Protein (230 series).jpg
- Cy5-labeled nucleic acids (nano.jpg)
