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PCR Related

Can you pool samples for PCR testing MNV?
Yes. Our PCR assay for MNV allows you to pool up to 10 samples with no loss of sensitivity.

What is the optimal sample for MPV testing by PCR?
Mesenteric lymph node has historically been used as the optimal target tissue for MPV PCR testing. However, recent studies by RADIL documented similar sensitivity for detection of MPV using spleen.  Recognizing that spleen is easier to locate and faster to collect, we recommend submitting a small piece of spleen for MPV PCR analysis.

We have a cell line that's Mycoplasma positive, but we need identification of the Mycoplasma. Does RADIL offer this service? If so, is the sample requirement the same as for Mycoplasma testing by PCR?
Yes, we do.  The method we use to identify the Mycoplasma sp. in cell cultures is to run our Mycoplasma sp. PCR assay on the sample.  We then sequence the PCR product and compare the sequence to genome sequences of other Mycoplasma sp. in GenBank.  This usually gives us a good ID of the contaminating Mycoplasma. 

The cost of performing this service is $200.  The sample requirements are the same as for our Mycoplasma PCR testing.  When requesting this service, please specify "Mycoplasma Sp. PCR and Sequence Analysis."

 

SEROLOGY Related

Why did the RADIL switch primary serology testing from ELISA to MFI?
MFI offers many advantages over the traditional ELISA. Among them are: increased sensitivity and specificity, better reproducibility, faster throughput of samples, the ability to assay for up to 100 different antigens and control preparations in a single well (multiplexing), and most importantly to you, the ability to perform ALL primary testing with only  0.2 µl of undiluted serum.

What is MFI?
MFI stands for Multiplex Fluorescent Immunoassay.  The technology is based both on bead-based immunoassay and flow cytometry.  Each purified RADIL antigen or control preparation is covalently linked to one of 100 different types of polystyrene beads, which vary slightly in the intensity of their color.  If IgG antibody to a particular antigen is present, it will bind to the antigen on a specific bead and will then be detected by subsequent binding of goat anti-mouse antibody conjugated to a fluorochrome, R-phycoerythrin.  The reader channels single beads through a dual laser detector which simultaneously determines both the bead type by the internal dye combination and the fluorescent intensity associated with each individual bead.  The fluorescent intensity associated with each of 100 individual beads of each type are used in the determination of each MFI value.

How do MFI results compare to ELISA results?
The RADIL has performed comparative, side-by-side testing of thousands of individual results from hundreds of samples.  The overall correlation between MFI and ELISA is greater than 99.5% for both mouse and rat samples.  In general, MFI is more sensitive than ELISA and is less prone to false positive results.

How does MFI fit into the RADIL serology testing strategy?
MFI is the state-of-the-art primary screening assay for rodent serology.  The secondary confirmatory testing technique will continue to be the indirect fluorescent antibody (IFA) test.  RADIL has added Western blot (WB) as the tertiary testing technique for the ultimate in sensitivity and specificity.

How much serum will I need to submit for MFI testing?
MFI requires only 0.2 µl of undiluted serum (1.0 µl of 1:5 diluted serum) regardless of the number of tests requested.  This means that survival bled rodents can now be comprehensively tested.

To allow for potential secondary and tertiary confirmatory testing, we recommend that a minimum of 20 µl of undiluted  serum (100 µl of diluted serum) be submitted for each sample.  With the advent of MFI technology, insufficient serum volumes have become a thing of the past.

What kinds of positive serum samples were used to validate the performance of the MFI system?
Wherever available, dilutions of known positive serum for mice and rats were used as the "gold standard" to assure assay performance.  In addition to this type of sample, we also used samples from experimentally infected rodents and clinical samples from well-characterized outbreaks to validate the performance of MFI.

How will you control for non-specifically adherent serum samples using MFI?
We have carefully chosen five control preparations that represent a wide spectrum of proteinaceous materials and we have established statistical parameters for determining if a given serum sample binds non-specifically to one or more of these preparations.  If that occurs and we also detect binding to antigens, we will report a result of NA for that particular antigen, just as we currently do in ELISA.  Non-specific binding will always occur to some extent in serologic testing.  However, it has been our experience that MFI is less prone to this problem than ELISA.  Of 30 mouse samples which yielded NA by ELISA and/or NF by IFA for two or more tests, 28 samples (93%) yielded valid results using the new MFI technology.  Thus, we anticipate MFI will yield a higher percentage of testable serum samples.

How were the baselines for MFI calculated?
Over 600 random negative mouse samples and 275 random negative rat samples (as determined by primary ELISA and secondary/tertiary testing as required) representing over 21,000 individual data points for all the agents and controls were used to calculate baseline values for MFI.  The baselines were set at the mean plus 5 standard deviation units for each agent / species combination and rounded off to the nearest 25 MFI units.  For example, the mouse MHV MFI baseline was calculated to be 373 and then rounded up to 375 (n=638).  Any value under 375 will be reported as negative, any value between 275 and twice that value, 750, will be reported as borderline (*) and automatically retested by IFA, while any value greater than 750 will be reported as positive. (+ to +33).

While mean positive to negative ratios of ELISA-tested samples are typically about 10:1, mean MFI positive to negative ratios are generally 50:1 and greater.  This means that from a statistical standpoint, it is much easier to differentiate between the negative and positive signals on MFI versus ELISA.  Thus, MFI testing should yield fewer equivocal results than traditional ELISA.

 

ANIMAL Related

When the weather is too cold to ship animals, is there a way to continue our health-monitoring and diagnostic  testing?
Yes.  Our Collect & Send services allow you to test without the concern or expense of shipping animals, and can be customized depending on your needs.  For more information on Collect & Send, click here.

 

 GENETICS Related

Both microsatellites and SNPS are available for testing of mice for genetic contamination.  Which is  the optimal approach?
We have made a conscious decision to use microsatellite technology versus single-nucleotide polymorphism (SNP) technolgy because microsatellites offer several distinct advantages.  Simple sequence length polymorphisms (SSLPs) or microsatellites are DNA regions containing di-, tri- or tetra-nucleotode repeats that are found randomly yet abundently throughout the genome.  The number of these repeats is highly polymorphic among different strains of mice and rats allowing them to serve as genetic markers that can distinguish between DNA derived from different strains.  In addition, micosatellites can be quickly and effciently assayed by PCR-based approaches making them a highly effective tool for genetic monitoring. 

SNPs on the other hand, are single base pair changes that  are found abundenlty throughout the genome although they may not be entirely randomly distributed in the mouse.  They are typically biallelic, meaning only two alleles tend to exist and thus are less informative than microsatellites which commonly have multiple alleles among different strains. It requires more SNPs to obtain the same genetic information as can be derived from microsatellites. Thus a major advantage of using microsatellites for genetic monitoring is the ability to readily detect genetic changes with a relatively small number of markers.  Additionally, when these changes are due to genetic contamination, it is often possible to identify the strain which is the source of the contamination. This is often much more difficult to do with SNPs.

 

CRYOPRESERVATION & REDERIVATION Related

I have a line of transgenic mice on a C57Bl/6 background that I used for some studies, but these studies have now been completed.  Therefore, I no longer need to maintain a colony of live animals.  I am thinking of cryopreserving this line in case I need to repeat some experiments or decide to utilize the line in future research projects.  Would it be best to freeze embryos or sperm? 
If the major goal is to cryopreserve the line for potential use at some undetermined time in the future, we recommend cryopreservation of sperm.  It is far less expensive to cryopreserve sperm ($600/line) and requires only 3-5 adult male mice.  In contrast, cryopreservation of embryos is more expensive ($2350/line) and requires 10-20 four- to six-week old female mice.  Please call to discuss your specific needs with one of our cryopreservation experts.