FGFR RGQ RT-PCR Kit

Paraffin-embedded or fixed samples typically yield fragmented, partially degraded RNA. In addition, RNA quality will depend greatly on the handling of the samples before, during, and after the fixation procedure. 

If performing RT-PCR with degraded RNA, we recommend using gene-specific primers or random nonamers rather than oligo-dT primers, since the mRNA poly-A tail may have been lost due to degradation.

We recommend that the RNeasy FFPE or miRNeasy FFPE kit be used to isolate the RNA.

RNA purified with the QIAamp Viral RNA Mini Kit has been used for quantification by qPCR with QuantiTect Probe RT-PCR Kit on QIAGEN Rotor-Gene Q.

The MinElute spin columns included in the following kits should be stored at 2–8°C upon arrival: AllPrep DNA/RNA Micro, EpiTect Fast DNA Bisulfite, EpiTect Fast FFPE Bisulfite, EpiTect Fast LyseAll Bisulfite, EpiTect Plus DNA Bisulfite, EpiTect Plus FFPE Bisulfite, EpiTect Plus LyseAll Bisulfite, exoRNeasy Serum/plasma Maxi, exoRNeasy Serum/Plasma Midi, GeneRead DNA FFPE, GeneRead rRNA Depletion, GeneRead Size Selection, MinElute Gel Extraction, MinElute PCR Purification, MinElute Reaction Cleanup, miRNeasy FFPE, miRNeasy Micro, miRNeasy Serum/Plasma, QIAamp DNA FFPE, QIAamp DNA Investigator, QIAamp DNA Micro, QIAamp MinElute Media, QIAamp MinElute Virus Spin, QIAamp MinElute Virus Vacuum, RNeasy FFPE, RNeasy Micro, RNeasy Plus Micro.

Short-term storage (up to 4 weeks) at room temperature (15–25°C) does not affect the performance. However, for optimal performance and quality, storage temperature should not exceed 25°C.

The RNeasy 96 Universal Tissue Kit enables high-throughput purification of RNA from any animal or human tissue sample, including difficult-to-lyse fibrous and fatty tissues. For single tube format RNA purification, the RNeasy Plus Universal Kit is also available. 

Please refer to the Selection guide for RNA isolation for all sample types to find the optimal solution for your sample source.

The exact composition of Buffer RLT is confidential. This buffer is a proprietary component of RNeasy Kits. Buffer RLT contains a high concentration of guanidine isothiocycanate, which supports the binding of RNA to the silica membrane. Buffer RLT can be purchased separately (cat. no. 79216)

Note: note that ß-mercaptoethanol should be added to Buffer RLT before use to effectively inactivate RNAses in the lysate (10 µl ß-Mercaptoethanol per 1 ml Buffer RLT).

The integrity and size distribution of total RNA purified with RNeasy Kits can be checked by denaturing-agarose gel electrophoresis, the Agilent 2100 bioanalyzer, or the QIAxcel Advanced System with the QIAxcel RNA QC Kit v2.0.

The respective ribosomal species should appear as sharp bands on the stained gel. 28S ribosomal RNA bands should be present with an intensity approximately twice that of the 18S RNA band. If the ribosomal bands are not sharp, but appear as a smear of smaller sized RNAs, it is likely that the RNA sample has suffered major degradation during preparation.

Size of ribosomal RNAs from various sources

Rotor-Gene Probe Kits are specially developed for Rotor-Gene cyclers. The unique rotary system of the cyclers combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Probe Kits do not contain ROX dye.

Both types of tubes run on the Rotor-Gene Q, and can be labeled on the top without any interference with data acquisition, because signal is detected from the bottom of the tube. Labeling is helpful and can prevent possible sample mix up.

There are several features available to allow exporting data across platforms to a variety of software packages. Sample names can easily be imported into the Rotor-Gene Q Excel sample sheets. By using a function "Export to Excel" the software automatically exports any results table in the software to Excel. Furthermore, reports can now be exported as Word files, which make the data compatible with any Microsoft office/Mac application. Reports can also be saved or sent in HTML format. All figures and graphs can be exported as Jpeg or Bitmap files for use in any desktop publishing software.

No. The master mixes in Rotor-Gene Kits contain dTTP instead of dUTP. If UNG treatment is required, we recommend using QuantiTect +UNG Kits. QuantiTect Kits are also compatible with the Rotor-Gene Q; however, the kits require a significantly longer cycling time.

No. The fixed optical path ensures uniform illumination and detection from sample to sample eliminating the need to use a reference dye such as ROX on the Rotor-Gene Q.

Yes. The sample sheet can be amended during or after a PCR run by activating the 'Edit samples' button in the Rotor-Gene Q software. The sample sheet will open and may be modified.

Yes, data can be analyzed on the Rotor-Gene Q while a run is in progress, allowing to save time for planning and setting up new experiments. Multiple copies of the Rotor-Gene Q software can be opened simultaneously during a run allowing analysis of previous experiments while the system is running.

QuantiTect Primer Assays are primer pairs designed and bioinformatically validated specifically for real-time RT-PCR with SYBR Green detection. To find a primer assay for your target gene of interest, please visit our GeneGlobe data base.

For best results, we strongly recommend using QuantiTect Primer Assays in combination with QIAGEN's products for SYBR Green-based Real-Time PCR and RT-PCR.

Please make data are collected in the appropriate fluorescent channel. Also check the gain is optimized.

If the issue persists, please send the original run file with extension .rex to QIAGEN Technical Service for further assistance.

The reaction volume to be entered in the Rotor-Gene Q software should be the sum of the PCR reaction volume and the volume of Vapor-Lock.

At the selected data acquisition step of the thermal cycle on the Rotor-Gene Q, the fluorescent signal intensity is measured 20 times for each sample as they spin past the detector. Tubes on a rotor spin past the excitation/detection optics every 150 milliseconds. The average of the 20 readings is then taken as the fluorescence of the sample at that cycle number.

Please send the original OTV run file to QIAGEN Technical Service for further assistance.

This type of cycling allows a significant reduction in cycling time for Rotor-Gene SYBR Green PCR Kits. It is more effective than reducing the individual times for annealing and extension.

We strongly recommend use of the dedicated Rotor-Gene Q Accessories, e.g., PCR Tubes 0.2 ml or Strip Tubes and Caps 0.1 ml provided by QIAGEN. These tubes are designed to give low background and perfectly match the Rotor-Gene Q requirements.

The buffer composition, which affects the initial reactivation of HotStarTaq Plus DNA Polymerase, has been optimized for each respective Rotor-Gene Kit.

A maximum of 5 µl RNA eluate from RNeasy extraction procedures can be added to the reverse-transcription reaction with the QuantiTect Whole Transcriptome Kit.

Yes, please choose from the Supplementary Protocols below:

QuantiFast Probe RT-PCR Kit:

• ABI PRISM 7000 Software Setup for the QuantiFast Probe RT-PCR Kit (PCR94)
• ABI PRISM 7700 Software Setup for the QuantiFast Probe RT-PCR Kit(PCR95)
• ABI PRISM 7900 Software Setup for the QuantiFast Probe RT-PCR Kit (PCR96)

QuantiFast Probe RT-PCR +ROX Vial Kit:

• Applied Biosystems 7500 Software Setup for the QuantiFast Probe RT-PCR +ROX Vial Kit (PCR97)
• DNA Engine Opticon Software Setup for the QuantiFast Probe RT-PCR +ROX Vial Kit (PCR98)
• iCycler iQ Software Setup for the QuantiFast Probe RT-PCR +ROX Vial Kit (PCR99)
• LightCycler 1.x Software Setup for the QuantiFast Probe RT-PCR +ROX Vial Kit (PCR100)
• LightCycler 2.0 Software Setup for the QuantiFast Probe RT-PCR +ROX Vial Kit (PCR101)
• LightCycler 480 Software Setup for the QuantiFast Probe RT-PCR +ROX Vial Kit (PCR102)
• Mastercycler ep realplex Software Setup for the QuantiFast Probe RT-PCR +ROX Vial Kit (PCR103)
• Mx3005P Software Setup for the QuantiFast Probe RT-PCR +ROX Vial Kit (PCR104)
• Rotor-Gene Software Setup for the QuantiFast Probe RT-PCR +ROX Vial Kit (PCR105)

The Rotor-Gene Q instrument can be used with two different rotor formats: using tubes or rotor-dics. Tubes can be run in 36 and 72-well rotors and rotor-discs in Rotor-Disc 72 and Rotor-Disc 100 rotors. When programming the temperature profile please make sure the correct rotor type is selected.  

Yes, use the assays at a final concentration of 1x with Rotor-Gene Probe Kits on the Rotor-Gene Q cycler.

See trademarks.  

Our unique multiplex PCR buffer system with ammonium and potassium ions and Factor MP has been further optimized in QuantiFast and Rotor-Gene Kits. We have also discovered Q-Bond, a buffer component which supports the rapid formation of the polymerase–primer–template complex, leading to reduced annealing times.

Our RT² qPCR Primer Assays may be used on any real-time instrument. qPCR solutions are available for the most popular qPCR instrumentation, including those from QIAGEN, ABI, BioRad, Stratagene.

Instrument-specific protocols are available for selected instruments, and can be accessed at the following link: http://www.sabiosciences.com/pcrarrayprotocolfiles.php

Based on the defined standards present, the automatic threshold function of the Rotor-Gene Q scans through all the possible threshold levels until the best fit is determined. This is defined as the R value or correlation coefficient, which is maximized to most closely approach 1.0. If there is no standard present, the threshold can also be set manually.

No, that is not necessary. Simply use the primer concentrations specified in the protocols in the handbook supplied with your Rotor-Gene Multiplex PCR Kit.

Yes. If the reaction efficiency between two PCR runs is not expected to vary, importing a standard curve from a previous run allows to estimate concentrations when a standard curve for the current run is not available. Curves can be imported from another channel, or from another run by clicking on 'Import Curve' in the Rotor-Gene Q software. Standard curves can be adjusted such that only the efficiency of the source curve is imported into the current run. Whether a standard curve should be adjusted depends on the PCR chemistry used. To adjust a standard curve, use a reference with a known concentration in the target run.

The majority of the Rotor-Gene Kit data shown in our literature has been generated with the help of the QIAgility instrument. We did not observe any problems during the pipetting steps.

The Rotor-Gene Q software version v2.2 and lower versions require a computer with Windows XP or Vista operating systems. The Rotor-Gene Q software v2.3 or higher versions can run with Windows XP or Windows 7 32-bit and 64-bit operating systems.

Rotor-Gene 6000 software requires a computer with Windows XP or Vista operating system. QIAGEN will no longer support Rotor-Gene 3000 instruments and Rotor-Gene 6.0 software starting from January 1, 2014

Small amounts of RNA and DNA may be difficult to measure spectrophotometrically. Fluorometric measurements, or quantitative RT-PCR and PCR are more sensitive and accurate methods to quantify low amounts of RNA or DNA.

Fluorometric measurements are carried out using nucleic acid binding dyes, such as RiboGreen® RNA Quantitation Reagent for RNA, and PicoGreen® DNA Quantitation Reagent for DNA (Molecular Probes, Inc.).

Yes, simply use the assays at a final concentration of 1x with the Rotor-Gene Multiplex PCR Kit.

See trademarks. 

Vapor-Lock is fully compatible with the QIAgility instrument for high-precision, automated reaction setup. It is also highly suited for use with the Rotor-Gene Q cycler. For support to program your QIAgility instrument for use with Vapor-Lock, please contact your local QIAGEN Technical Support Department.

Tissue-Tek O.C.T. is an embedding compound for cryosectioning, which is soluble in water. It mainly consists of glycols and synthetic resins. Tissue-Tek O.C.T. is used as matrix for cryosectioning of tissues. Using the O.C.T. the tissue samples can be positioned more easily in the microtome and have better qualities during sectioning.

Most cryosections are fixed using non-crosslinking agents. For isolation of RNA from tissue embedded in Tissue-Tek O.C.T. using non-crosslinking agents the RNeasy Plus Micro Kit or the RNeasy Micro Kit (Protocol: Total RNA Isolation from Microdissected Cryosections), or the RNeasy Mini Kit (RNeasy Mini Protocol for the isolation of Total RNA from Animal Tissue) give great results. We strongly recommend removing as much of the embedding compound as possible prior to RNA extraction from the sections. Please see QIAGEN News article, Issue 1 1998, 'Effects of malnutrition on expression of lactase in children' for successful RNA isolation from O.C.T.-embedded tissue using the RNeasy Mini Kit.

In case crosslinking agents (e.g. formaldehyde or glyoxal-containing) were used for fixation of the tissue for cryosectioning the RNeasy FFPE Kit is the perfect choice. The RNeasy FFPE Kit is especially designed for purifying total RNA from formalin-fixed tissue sections. Special lysis and incubation conditions reverse formaldehyde modification of RNA for improved results in downstream application. The crosslinking causes the RNA to break, resulting in overall smaller molecules, which look like a smear when analyzed on a formaldehyde gel.

In case DNA as well as RNA from precious samples is of interest from the identical sections, please have a look at the AllPrep DNA/RNA Kits for unfixed tissue or the AllPrep DNA/RNA FFPE Kit for fixed tissue.

Use of endogenous control genes corrects for variation in RNA content, variation in reverse-transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA sample, variation in nucleic acid recover, and differences in sample handling. The endogenous control gene ought to have consistent expression levels between samples and among treatment conditions, and ideally has a similar expression level to that of the genes of interest. Genes commonly used as references can be found at the QuantiTect Primer Assays as endogenous controls.

We have performed numerous tests comparing the performance of Rotor-Gene SYBR Green PCR Kits and QuantiTect Kits with QuantiTect Primer Assays. Due to the optimized ion concentrations in the PCR buffers, both perform equally well with QuantiTect Primer Assays and do not require any adjustment of primer concentration.

The efficiency of downstream applications depends strongly on the purity of the RNA sample used.  Pure RNA should yield an A260/A230 ratio of around 2 or slightly above; however, there is no consensus on the acceptable lower limit of this ratio.  Possible candidates that can increase the A230 include “salt”, carbohydrates, peptides, and phenol (or aromatic compounds in general).  In our experience, the increased absorbance at 230 nm in RNA samples is almost always due to contamination with guanidine thiocyanate, present at very high concentrations in the lysis buffer or extraction reagent used in most RNA purification procedures.

Please find an article discussing the effect of low 260/230 ratios in RNA preparations on downstream applications on page 7 of QIAGEN Newsletter March 15, 2010 . In summary, we found that concentrations of guanidine thiocyanate of up to 100 mM in an RNA sample do not compromise the reliability of downstream applications.

Rotor-Gene Kits are specifically developed for the Rotor-Gene Q PCR Cycler. The unique rotary system of the cycler combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Kits do not contain ROX dye since no normalization to a passive reference is required. Also, Rotor-Gene Kits do not contain dUTP; therefore, UNG pretreatment is not possible.

To ensure efficient gDNA removal when doing an on-column digest using the RNase-Free DNase Set in combination with RNeasy Mini the following factors are crucial:

• prevent overloading by adjusting the amount of starting material to no more than the maximum amounts recommended in the RNeasy Mini Handbook
• ensure complete disruption and homogenization of the starting material as instructed in the section 'Disruption and homogenization of starting materials' of the handbook
• strictly follow the protocol for on-column DNase Digestion in Appendix D of the RNeasy Mini Handbook (you can let wash buffer RW1 incubate on the column for 3-5 minutes before centrifuging to enhance removal of excess gDNA prior to applying the enzyme)

In the rare case that trace amounts of genomic DNA are still detectable in sensitive downstream applications such as e.g., realtime RT-PCR, an in-solution digest using the RNase-Free DNAase set can be performed. Instructions are presented in Appendix C of the RNeasy MinElute Cleanup Handbook.

Alternatively, a second on-column digest can be carried out in future preparations, immediately following the RW1 wash after the first incubation with DNase.

No. The speed at which the samples spin on the Rotor-Gene Q is not high enough to apply any significant centrifugal force on them.

The sample rotor of the Rotor-Gene Q spins continuously at a speed of 400 rpm during a run.

The specific features of Rotor-Gene Kits and Rotor-Gene cyclers work synergetically to enable an ultrafast-cycling protocol. We do not guarantee that the performance of the Rotor-Gene Multiplex RT-PCR Kit with the same cycling protocol will be the same on other cyclers.

Yes. Rotor-Gene Kits will also work on the Rotor-Gene 6000 and Rotor-Gene 3000 PCR cyclers with the cycling conditions specified in the Rotor-Gene kit handbooks.

The exact composition of Buffer RW1 is confidential. Buffer RW1 is a proprietary component of RNeasy Kits. Buffer RW1 contains a guanidine salt, as well as ethanol, and is used as a stringent washing buffer that efficiently removes biomolecules such as carbohydrates, proteins, fatty acids etc., that are non-specifically bound to the silica membrane. At the same time, RNA molecules larger than 200 bases remain bound to the column.

Note: Buffer RW1 should not be used for isolation of small RNAs, for example, microRNAs or fragmented RNA from formalin-fixed tissues, as these smaller fragments will be washed away.  Buffer RWT should be used instead.