QIAGEN RNeasy User Manual
April 2021
RNeasy® Protect Saliva
Mini Handbook
For immediate stabilization of total RNA in saliva and subsequent RNA purification
Sample to Insight__
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Contents
Kit Contents .................................................................................................................. 3
Storage ..................................................................................................................... 4
Intended Use .............................................................................................................. 4
Safety Information ....................................................................................................... 5
Quality Control ........................................................................................................... 5
Introduction ................................................................................................................ 6
Principle and procedure .................................................................................... 6
Equipment and Reagents to Be Supplied by User ............................................................ 9
Protocol: Stabilization of RNA in Saliva using RNAprotect Saliva Reagent ....................... 10
Protocol: Purification of RNA from Stabilized Saliva using the RNeasy Micro Kit .............. 12
Troubleshooting Guide .................................................................................................. 17
Appendix A: General Remarks on Handling RNA......................................................... 20
Appendix B: Storage, Quantification and Determination of Quality of RNA ..................... 23
Ordering Information ................................................................................................ 27
Document Revision History ......................................................................................... 33
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Kit Contents
RNeasy Protect Saliva Mini Kit
Catalog no
Number of preps
RNAprotect Saliva Reagent (box 1 of 2):
RNAprotect® Saliva Reagent 50 ml
RNeasy Protect Saliva Mini Handbook* 1
RNeasy Micro Kit (box 2 of 2):
RNeasy MinElute® Spin Columns
(each in a 2 ml Collection Tube) 50
Collection Tubes (1.5 ml) 50
Collection Tubes (2 ml) 100
Buffer RLT† 45 ml
Buffer RW1† 45 ml
Buffer RPE‡ (concentrate) 11 ml
RNase-Free Water 10 ml
Carrier RNA, poly-A 1 vial (300 µg)
RNase-Free DNase Set
RNase-Free DNase I (lyophilized)
Buffer RDD
RNase-Free Water
Handbook* 1
* Follow the instructions in the RNeasy Protect Saliva Mini Handbook when stabilizing and purifying RNA from saliva.
†
Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 5 for safety information.
‡
Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a working
solution.
(50)
74324
50
1500 units
2 x 2 m
1.5 ml
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Storage
Store RNAprotect Saliva Reagent at room temperature (15–25°C). The reagent is stable for at
least 12 months under these conditions, if not otherwise stated on the label.
The RNeasy Micro Kit is shipped at room temperature. Store the RNeasy MinElute spin columns
and the RNase-Free DNase Set box (containing RNase-free DNase, Buffer RDD and RNasefree water) immediately upon receipt at 2–8°C. Store the remaining components of the RNeasy
Micro Kit dry at room temperature. All components of the RNeasy Micro Kit are stable for at
least 9 months under these conditions, if not otherwise stated on the label.
Intended Use
The RNeasy Protect Saliva Mini Kit is intended for molecular biology applications. This product
is not intended for the diagnosis, prevention or treatment of a disease. All due care and
attention should be exercised in the handling of the products. We recommend all users of
QIAGEN® products to adhere to the NIH guidelines that have been developed for recombinant
DNA experiments or to other applicable guidelines.
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Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, please consult the appropriate safety data sheets
(SDSs). These are available online in convenient and compact PDF format at
www.qiagen.com/safety where you can find, view and print the SDS for each QIAGEN kit
and kit component.
CAUTION
CAUTION: DO NOT add bleach or acidic solutions directly to the sample
preparation waste.
Buffer RLT contains guanidine thiocyanate and Buffer RW1 contains a small amount of
guanidine thiocyanate. This chemical can form highly reactive compounds when combined
with bleach. If liquid containing these buffers is spilt, clean with suitable laboratory detergent
and water. If the spilt liquid contains potentially infectious agents, clean the affected area first
with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of RNeasy
Protect Saliva Mini Kit is tested against predetermined specifications to ensure consistent
product quality.
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Introduction
The RNeasy Protect Saliva Mini procedure provides a complete solution for the stabilization
and purification of total RNA from saliva. RNA in collected saliva is immediately stabilized
using RNAprotect Saliva Reagent, and then rapidly purified and concentrated using the
RNeasy Micro Kit.
Principle and procedure
RNA stabilization using RNAprotect
Saliva Reagent Immediate stabilization of RNA in a saliva sample is a prerequisite for reliable
gene expression analysis using microarray, real-time RT-PCR or other nucleic-acid-based
technology. This is because changes in the gene expression pattern occur immediately after
saliva collection due to unspecific and specific RNA degradation as well as to transcriptional
induction. Also, since saliva contains bacteria in addition to cells and free-circulating molecules
(e.g., nucleic acids) of human origin, it is important to prevent the uncontrolled growth of
bacteria, which can also affect the gene expression profile. RNAprotect Saliva Reagent uses
a novel patent-pending technology to immediately stabilize the gene expression profile in
saliva samples, which can then be stored and transported at ambient temperature.
Note: RNA in saliva is already degrading prior to collection and stabilization with RNAprotect
Saliva Reagent. This means that RNA purified from saliva samples will be of lower quality than
RNA purified from other samples types, such as cultured cells and animal tissues.
RNA purification using the RNeasy Micro Kit
RNeasy Micro technology combines the selective binding properties of a silica–based
membrane with the speed of microspin technology. Guanidine-thiocyanate–containing lysis
buffer and ethanol are added to the sample to create conditions that promote selective binding
of RNA to the RNeasy MinElute membrane. The sample is then applied to the RNeasy MinElute
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spin column. RNA binds to the silica membrane. Traces of DNA that may copurify are removed
by a DNase treatment on the spin column. DNase and any contaminants are washed away,
and high-quality total RNA is eluted in RNase-free water (see flowchart, next page).
With the RNeasy Micro procedure, all RNA molecules longer than 200 nucleotides are
purified. The procedure enriches for mRNA since most RNAs <200 nucleotides (such as 5.8S
rRNA, 5S rRNA and tRNAs, which together make up 15–20% of total RNA) are selectively
excluded. The size distribution of the purified RNA is comparable to that obtained by
centrifugation through a CsCl cushion, where small RNAs do not sediment efficiently.
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Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, consult the appropriate safety data sheets (SDSs),
available from the product supplier.
For saliva collection and RNA stabilization
Ice
Vessels for collecting saliva (e.g., 50 ml polypropylene tubes)
2 ml microcentrifuge tubes
Vortexer
For RNA purification
Ethanol (70% and 96–100%)*
Sterile, RNase-free pipet tips
Microcentrifuge (with rotor for 2 ml tubes)
Vortexer
Disposable gloves
* Do not use denatured alcohol, which contains other substances, such as methanol or methylethylketone.
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Protocol: Stabilization of RNA in Saliva using
RNAprotect Saliva Reagent
Important points before starting
The subject should refrain from eating, drinking or oral hygiene procedures for at least
1 hour prior to collection of saliva. Optionally, the mouth can be rinsed with water
(without swallowing) 5 minutes before saliva collection.
Collect saliva using a collection vessel with an appropriate volume and a sufficiently
large opening (e.g., a 50 ml polypropylene tube). The subject should spit into the
collection vessel, but should not cough up mucus.
During saliva collection, keep the collection vessel on ice to minimize changes in the
gene expression pattern. After saliva collection, the sample should be immediately mixed
with RNAprotect Saliva Reagent. RNA is not stabilized until the sample is treated with
RNAprotect Saliva Reagent.
Perform the procedure described below as quickly as possible.
Procedure
1. Collect 200 µl saliva in a collection vessel placed on ice. Proceed immediately to step If
processing larger volumes of saliva, collect several samples of 200 µl saliva.
Note: The RNA in the saliva sample is not stabilized until the sample is treated with
RNAprotect Saliva Reagent in step 2.
2. Add 200 µl saliva to 1 ml RNAprotect Saliva Reagent in a 2 ml microcentrifuge tube (not
supplied). Mix well by vortexing.
The RNA in the saliva sample is now stabilized.
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3. Store the mix of saliva and RNAprotect Saliva Reagent for up to 1 day at 37°C, up to
14 days at room temperature (15–25°C), or up to 28 days at 2–8°C, or archive at –
90°C to ––15°C. 0.
Note: We recommend lower storage temperatures whenever possible (e.g., 2–8°C
instead of room temperature or room temperature instead of 37°C).
Note: A precipitate may form during storage, especially at lower temperatures. This does
not affect RNA purification.
Note: To ensure maximal RNA yields, store the stabilized saliva sample for at least
24 hours before starting RNA purification. If the stabilized samples have been frozen at 95°C or -15°C before extraction, this incubation step can be skipped.
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