Frequently Asked Questions

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Once stored in this buffer, can I use the RNA for cDNA prep followed by qPCR or one step qPCR or one step RT-ddPCR directly?

At present, this guidance is based on Plasmidsaurus RNA-seq processing, as this is the context in which the product has been evaluated. If alternative downstream methods are used, performance should be validated independently.

RNA stability is also influenced by the quality of sample extraction and purification. High-quality RNA preparations (i.e., low RNase contamination and minimal metal ion presence) are associated with improved stability over time in SEQguard, whereas lower-quality preparations may result in faster degradation.

RNA should be eluted in a low-EDTA TE buffer (10 mM Tris, 0.1 mM EDTA), such as IDTE buffer (IDT, pH 7.5).

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On Plasmidsaurus website, it’s indicated that: Mix 24 μL of RNA in IDTE buffer with 6 μL of SEQguard Dino Preserve to achieve a final concentration between 10-100 ng/μL. If the RNA is eluted in nuclease-free water – can I still mix it with SEQguard at this ratio?

Samples can be submitted to Plasmidsaurus in either nuclease-free water supplemented with SEQguard Dino Preserve (mixed at the same ratio recommended for IDTE buffer: 24 μL RNA with 6 μL SEQguard Dino Preserve) or in IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5).

Submission in IDTE buffer is recommended, as the addition of EDTA to a final concentration of up to 0.1 mM has been shown to improve RNA stability at elevated temperatures. Samples submitted in nuclease-free water will still be processed.

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Can you tell me how long RNA is stable in this buffer in ambient temperature, +4°C, – 20°C, and – 80°C?

Ambient temperature: RNA in SEQguard has been shown to maintain high quality for Plasmidsaurus library preparation for 7 days, often longer. However, shipping times should still be minimized. Typical transit times are routinely acceptable without issue.

+4°C: Provides longer stability than ambient temperature.

−20°C: Suitable for storage over weeks to months.

−80°C: Suitable for long-term storage (effectively indefinite).

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Can I use FabRICATOR (IdeS) with very low amounts of IgG?

It is possible to use FabRICATOR with low concentrations of IgG. Low concentrations of IgG will lead to decreased reaction rate, so for concentrations below 0.5mg/mL it is therefore recommended to increase the amount of added enzyme and/or increase the incubation time. There is no risk of over-digestion and the incubation time can be prolonged to O/N if necessary.

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I have a small modification in the sequence below the hinge, will FabRICATOR still digest my IgG?

FabRICATOR is a very specific enzyme and small changes in the sequence in or around the digestion site (…CPAPELLG / GPSVF…) may reduce or completely impair the ability to digest the IgG. It is very difficult to predict the outcome for each specific modification. It might work, but we cannot guarantee it unfortunately, the only way of knowing is to perform a digestion test. Some suggestions that are worth testing if you experience incomplete digestion: Increase the incubation time, increase the amount of added enzyme, use a PBS with greatly reduced salt concentration (5-10 mM instead of 140 mM) and pH 6.5 instead of 7.4.

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Can FabRICATOR be stored in frozen stock solutions?

We have no stability data for storage at -20°C or -70°C. However, we have customer reporting that they successfully stored reconstituted FabRICATOR at -20°C or -70°C for a couple of months. Genovis cannot guarantee the activity of the enzyme, but these storage conditions might be OK depending on the intended application.

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Is FabRICATOR stable in the presence of denaturing agents such as guanidine hydrochloride or SDS?

We have tested 4-6 M GuHCl which the enzyme does not withstand without significant decrease of activity. SDS have not been evaluated but 0.1% RapiGest from waters have negative influence on activity. Taken together the enzyme does not well tolerate denaturing agents although we have not tested this in more detailed studies.

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Does FabRICATOR introduce modifications?

FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (…CPAPELLG / GPSVF…) and the C-terminus will regain the carboxyl group – that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).

In summary, R1-CO-NH-R2 + H2O –> R1-COOH + NH2-R2.

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Does FabRICATOR (IdeS) have protease activity on IgM?

No. According to the following reference, FabRICATOR does not digest IgM. Pavel-Rammingen et al., 2002. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.

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Where does FabRICATOR cleave the IgG molecule?

FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab’)2 fragment and a Fc-fragment.
Human IgG1: ..CPAPELLG / GPSVF..
Human IgG2: ..CPAPPVA / GPSVF..
Human IgG3: ..CPAPELLG / GPSVF..
Human IgG4: ..CPAPEFLG / GPSVF..

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