Determination of HEGA filter size
Two factors are required to determine the proper size of the filter: (1) the maximum supply air flow rate and (2) the maximum pressure drop across the filter. Generally, the maximum supply air flow rate of a particular laboratory can be calculated by determining the maximum flow-rate across the fume-hood(s). For example, a laboratory with a single 5 foot fume hood and a maximum sash opening height of 2 feet will create a sash opening area of 10 ft2. The total flow rate is the sash opening (10 ft2) multiplied by the fume hood minimum face velocity (100 ft/minute is typical for chemical fume hoods) or 1,000 ft3/minute. To determine the maximum pressure drop across the HEGA filter, maximize the air flow rate to the laboratory by opening the fume hood sash and lowering the thermostat. If the damper is wide open, that is an indication that there is not enough static pressure in the duct to overcome the additional resistance of the HEGA filter without modifying the fan speed or increasing the size of the duct or dampers. If the damper is not fully open, there is potentially enough pressure in the duct to overcome the added resistance of a new HEGA filter. The maximum pressure available can be determined by the following process. (1) Measure the static pressure at the location of the proposed HEGA filter. (2) Add resistance to the duct at the supply air diffusers until the supply damper is fully open. (3) Measure the static pressure reading at the location of the proposed HEGA filter under these conditions. The difference between the two readings will determine the maximum pressure permitted across the new HEGA filter.
Carbon-filtration of laboratory environment
The process for adding carbon filtration to our existing HVAC system involved several steps. Using the maximum supply air flow rate and the maximum pressure drop across the filter as described above, National Center for Toxicological Research engineers selected an appropriate carbon loaded non-woven filter. The HEGA filter series 2653 (part number 11–17979; Filtration Group, Joliet, IL, USA) which is a 24" × 24" × 12" carbon filter and which has a low pressure drop of 0.3 inches (water column) at the maximum air flow rate of 1,100 cubic feet per minute was used. Because of the low pressure drops, these gas phase carbon filters can be successfully installed in many high pressure systems provided that enough space exists above the ceiling for the filter housing.
Although the carbon filter prevented the vast majority of environmental ozone from entering the laboratory, without the laboratory air pressure being positive with respect to adjacent spaces, ozone-contaminated air may leak in around door frames and the unsealed perimeter of the laboratory. In both variable air volume (VAV) and constant air volume (CAV) HVAC systems, the exhaust box is designed to track the supply air flow with a typical adjustable offset of 50 to 100 ft3/minute. For example, when the exhaust VAV box tracks the supply VAV box with a positive offset of 100 ft3/minute and the supply is 1,000 ft3/minute, the exhaust will be 1,100 ft3/minute. The 100 ft3/minute offset will make the laboratory negatively pressurized with respect to its adjacent spaces and 100 ft3/minute of air from the surrounding areas would infiltrate the lab. The HEGA filter is effective at removing ozone to 2 ppb or less. If the ozone level in the surrounding area is 50 ppb, the laboratory would contain a mixture of 1,000 ft3/minute at 2 ppb ozone and 100 ft3/minute at 50 ppb. The overall ozone level would increase from 2 ppb to 6.4 ppb based on the following calculation: (1,000/1,100) × 2 + (100/1,100) × 50 = 6.4 ppb. To eliminate this increase in ozone and maintain the ozone level at approximately 2 ppb, the laboratory must be positively pressurized with respect to the hallway.
In-house produced microarrays
In-house printed microarrays were constructed using the mouse 20,000 oligonucleotide set and the rat 10,000 oligonucleotide from MWG (High Point, NC, USA). The oligonucleotides were dissolved in 1× MWG Spotting Buffer A at a concentration of 20 μM and printed on poly-L-lysine-coated slides (Erie Scientific, Portsmouth, NH) using an OmniGrid Microarrayer (GeneMachines, San Carlos, CA). Printed slides were processed and stored in a desiccator at room temperature before use [9].
Target cDNAs were labeled with cyanine dyes using cDNA indirect labeling protocol [9]. Cy3- and Cy5- labeled cDNAs were mixed together and concentrated to a volume of less than 5 μl using a SpeedVac SPD 1010 (Themo Savant, Holbrook, NY) at room temperature. The samples were then mixed with 60 μl of pre-warmed hybridization buffer. Detailed hybridization and washing procedures have been described previously [9].
Commercial microarrays
Microarrays (22K mouse oligonucleotides) manufactured by Agilent Technologies (Cat. No. G4121A) were hybridized according to Agilent protocols. Total RNAs were labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Cat. No. 5184-3523). The hybridization and washing procedures were also performed following the Agilent 60-mer Oligo Microarray Processing Procedure (Cat. No. G4140-90030).
Scanning, feature extraction, and data analysis
Immediately after the slide washing and spin-drying procedures, the microarrays were scanned using the Axon 4000B microarray scanner (Molecular Devices, Sunnyvale, CA, USA) with photomultiplier tube settings balanced for the 635 nm and 532 nm channels. Following the initial scans of paired microarrays in the carbon-filtered laboratory, each slide was placed in a separate black plastic 25-slide box with the lid ajar to permit free air flow and to block direct light from striking the microarray. One box was placed in the adjacent hallway in which the air is not carbon-filtered; the other box remained in the lab with the carbon filtered air. The hallway was used so that ozone measurements could be made in real-time in both the laboratory (reduced ozone) and the hallway (ambient ozone). Scans of each slide of a pair were alternated for the duration of the experiment. It should be noted that the slides placed in the hallway were only exposed to atmospheric ozone when placed in the hallway and that during scanning, by necessity, they were in the carbon-filtered (reduces ozone) environment. Thus the actual ozone exposure for these slides is approximately 1/2 of the total experiment time. Ozone levels were measured using a Model 450 Ozone Monitor (Advanced Pollution Instrumentation, Inc., San Diego, CA, USA).
The resulting images were analyzed by measuring the fluorescence of all features on the microarrays using the GenePix Pro 6.0 image analysis software (Molecular Devices, Sunnyvale, CA, USA). The median fluorescence intensity of all the pixels within one feature was taken as the intensity value for that feature. All the raw data were imported into ArrayTrack Software [10] and were normalized using LOWESS Normalization with background subtraction. The data correlation values were computed with JMP 6.0 software (SAS, Inc., Cary, NC).